Ekaterina V. Sheshukova

Metabolic Methanol: Molecular Pathways and Physiological Roles

Methanol has been historically considered an exogenous product that leads only to pathological changes in the human body when consumed. However, in normal, healthy individuals, methanol and its short-lived oxidized product, formaldehyde, are naturally occurring compounds whose functions and origins have received limited attention. There are several sources of human physiological methanol. Fruits, vegetables, and alcoholic beverages are likely the main sources of exogenous methanol in the healthy human body. Metabolic methanol may occur as a result of fermentation by gut bacteria and metabolic processes involving S-adenosyl methionine. Regardless of its source, low levels of methanol in the body are maintained by physiological and metabolic clearance mechanisms. Although human blood contains small amounts of methanol and formaldehyde, the content of these molecules increases sharply after receiving even methanol-free ethanol, indicating an endogenous source of the metabolic methanol present at low levels in the blood regulated by a cluster of genes. Recent studies of the pathogenesis of neurological disorders indicate metabolic formaldehyde as a putative causative agent. The detection of increased formaldehyde content in the blood of both neurological patients and the elderly indicates the important role of genetic and biochemical mechanisms of maintaining low levels of methanol and formaldehyde.

Cell wall methanol as a signal in plant immunity

Cell wall pectin forms a matrix around the cellulose–xyloglucan network that is composed of rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan (HG), a major pectic polymer consisting of α-1,4-linked galacturonic acids. HG is secreted in a highly methyl-esterified form and selectively de-methyl-esterified by pectin methylesterases (PMEs) during cell growth and pathogen attack. The mechanical damage that often precedes the penetration of the leaf by a pathogen promotes the activation of PME, which in turn leads to the emission of methanol (MeOH), an abundant volatile organic compound, which is quickly perceived by the intact leaves of the damaged plant, and the neighboring plants. The exposure to MeOH may result in a “priming” effect on intact leaves, setting the stage for the within-plant, and neighboring plant immunity. The emission of MeOH by a wounded plant enhances the resistance of the non-wounded, neighboring “receiver” plants to bacterial pathogens and promotes cell-to-cell communication that facilitates the spread of viruses in neighboring plants.

Endogenous Methanol Regulates Mammalian Gene Activity

We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.

Dietary Methanol Regulates Human Gene Activity

Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimers disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.

Pectin Methylesterase-Generated Methanol May Be Involved in Tobacco Leaf Growth

Plant leaves undergo a sink-source modification of intercellular macromolecular transport during the transition from carbon import to carbon export. After assessing the role of metabolite signaling in gene regulation in Nicotiana tabacum sink and source leaves, we observed increased pectin methylesterase (PME)-mediated methanol generation in immature leaves. Using suppression subtractive hybridization (SSH), we identified a number of genes whose activity changes from sink to source leaves. The most abundant SSH-identified genes appeared to be sensitive to methanol. We hypothesize that tobacco leaf maturation and the sink-source transition are accompanied by a change in mRNA levels of genes that function in methanol-dependent cell signaling.

Plant factories for the production of monoclonal antibodies

Like animal cells, plant cells bear mechanisms for protein synthesis and posttranslational modification (glycosylation and phosphorylation) that allow them to be seriously considered as factories for therapeutic proteins, including antibodies, with the development of biotechnology. The plant platform for monoclonal antibody production is an attractive approach due to its flexibility, speed, scalability, low cost of production, and lack of contamination risk from animal-derived pathogens. Contemporary production approaches for therapeutic proteins rely on transgenic plants that are obtained via the stable transformation of plant cells as well as the transient (temporary) expression of foreign proteins. In this review, we discuss present-day approaches for monoclonal antibody production in plants (MAPP), features of carbohydrate composition, and methods for the humanization of the MAPP carbohydrate profile. MAPPs that have successfully passed preclinical studies and may be promising for use in clinical practice are presented here. Perspectives on using MAPPs are determined by analyzing their economic benefits and production rates, which are especially important in personalized cancer therapy as well as in cases of bioterrorism and pandemics.

Volatile organic compounds and plant virus–host interaction

Abstract Plant cells are bounded by a dense cell envelope that prevents the penetration of viruses. Wounding of the cell wall (CW) through microdamage caused by abiotic factors (wind, hail, rain) and biotic factors (insects) may allow the penetration of virus particles into the cell. Once in the cytoplasm, the viral genome is translated, replicated, and later transferred to neighboring cells and throughout the plant. On the other hand, mechanical damage to the leaf promotes the emission of volatile organic compounds (VOCs), such as green leaf volatiles and methanol (MeOH), which are quickly taken up by the intact leaves of the damaged plant and the neighboring plants. The transport of VOCs is much faster compared to the transport of infectious viral entities through the phloem during systemic infection of a plant. Thus, exposure to VOCs may result in a ‘priming’ effect on intact leaves, setting the stage for the subsequent viral infection. In tobacco leaves, MeOH induces the synthesis of mRNA encoding the so-called methanol-inducible genes, most of which are associated with stress, defense, and the intercellular transport of macromolecules. The emission of MeOH by a wounded plant enhances the resistance of non-wounded neighboring ‘receiver’ plants to the bacterial pathogen Ralstonia solanacearum and promotes cell-to-cell communication that facilitates the spread of Tobacco mosaic virus in neighboring plants. For the vector-transmitted viruses, such as persistent viruses ( Potato leafroll virus and Barley yellow dwarf virus ) and non persistent viruses ( Cucumber mosaic virus ), VOCs also play a favorable auxiliary role, whereas VOCs are alarm cues that promote protective mechanisms in response to other pathogens (bacteria, fungi, oomycetes, nematodes) and herbivore attack. The different effects of VOCs on plant viruses and other pathogens seem to be due to differences in the localization of the pathogen. While a virus spends its entire life cycle in the cell symplast, bacteria and other pathogens reside in the apoplast. VOCs are apoplastic factors that are transferred through the air and affect the structure of the apoplast, including the CW, which is essential for plant immunity. Our knowledge of the role of VOCs in the viral life cycle and in inter- and intra-plant communication remains incomplete. When considering the use of VOCs for plant protection, we must keep in mind the potential sensitization of plants to viral infections, which is most likely the inevitable price of resistance to bacteria, fungi, oomycetes, and nematodes.

Functional role of carbohydrate residues in human immunoglobulin G and therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies (TMA) provide an important means for treating diseases that were previously considered untreatable. Currently more than 40 full-size TMAs created primarily based on immunoglobulin G1 are widely used for treating various illnesses. Glycosylation of TMA is among other numerous factors that affect their biological activity, effector functions, immunogenicity, and half-life in the patient’s serum. The importance of carbohydrate residues for activity of human serum immunoglobulin and TMA produced in animal cells is considered in this review, with emphasis given to N-glycosylation of the Fc fragment of the antibody.

The intergenic interplay between aldose 1-epimerase-like protein and pectin methylesterase in abiotic and biotic stress control

The mechanical damage that often precedes the penetration of a leaf by a pathogen promotes the activation of pectin methylesterase (PME); the activation of PME leads to the emission of methanol, resulting in a “priming” effect on intact leaves, which is accompanied by an increased sensitivity to Tobacco mosaic virus (TMV) and resistance to bacteria. In this study, we revealed that mRNA levels of the methanol-inducible gene encoding Nicotiana benthamiana aldose 1-epimerase-like protein (NbAELP) in the leaves of intact plants are very low compared with roots. However, stress and pathogen attack increased the accumulation of the NbAELP mRNA in the leaves. Using transiently transformed plants, we obtained data to support the mechanism underlying AELP/PME-related negative feedback The insertion of the NbAELP promoter sequence (proNbAELP) into the N. benthamiana genome resulted in the co-suppression of the natural NbAELP gene expression, accompanied by a reduction in the NbAELP mRNA content and increased PME synthesis. Knockdown of NbAELP resulted in high activity of PME in the cell wall and a decrease in the leaf glucose level, creating unfavorable conditions for Agrobacterium tumefaciens reproduction in injected leaves. Our results showed that NbAELP is capable of binding the TMV movement protein (MPTMV) in vitro and is likely to affect the cellular nucleocytoplasmic transport, which may explain the sensitivity of NbAELP knockdown plants to TMV. Although NbAELP was primarily detected in the cell wall, the influence of this protein on cellular PME mRNA levels might be associated with reduced transcriptional activity of the PME gene in the nucleus. To confirm this hypothesis, we isolated the N. tabacum PME gene promoter (proNtPME) and showed the inhibition of proNtPME-directed GFP and GUS expression in leaves when co-agroinjected with the NbAELP-encoding plasmid. We hypothesized that plant wounding and/or pathogen attack lead to PME activation and increased methanol emission, followed by increased NbAELP expression, which results in reversion of PME mRNA level and methanol emission to levels found in the intact plant.

An Alternative Nested Reading Frame May Participate in the Stress-Dependent Expression of a Plant Gene

Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP, a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is “matryoshka,” containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5′-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves.