Anastasia V. Shindyapina

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.

Methanol May Function as a Cross-Kingdom Signal

Recently, we demonstrated that leaf wounding results in the synthesis of pectin methylesterase (PME), which causes the plant to release methanol into the air. Methanol emitted by a wounded plant increases the accumulation of methanol-inducible gene mRNA and enhances antibacterial resistance as well as cell-to-cell communication, which facilitates virus spreading in neighboring plants. We concluded that methanol is a signaling molecule involved in within-plant and plant-to-plant communication. Methanol is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of methanol into toxic formaldehyde. However, recent data showed that methanol is a natural compound in normal, healthy humans. These data call into question whether human methanol is a metabolic waste product or whether methanol has specific function in humans. Here, to reveal human methanol-responsive genes (MRGs), we used suppression subtractive hybridization cDNA libraries of HeLa cells lacking ADH and exposed to methanol. This design allowed us to exclude genes involved in formaldehyde and formic acid detoxification from our analysis. We identified MRGs and revealed a correlation between increases in methanol content in the plasma and changes in human leukocyte MRG mRNA levels after fresh salad consumption by volunteers. Subsequently, we showed that the methanol generated by the pectin/PME complex in the gastrointestinal tract of mice induces the up- and downregulation of brain MRG mRNA. We used an adapted Y-maze to measure the locomotor behavior of the mice while breathing wounded plant vapors in two-choice assays. We showed that mice prefer the odor of methanol to other plant volatiles and that methanol changed MRG mRNA accumulation in the mouse brain. We hypothesize that the methanol emitted by wounded plants may have a role in plant-animal signaling. The known positive effect of plant food intake on human health suggests a role for physiological methanol in human gene regulation.

Mineralization of the Connective Tissue: A Complex Molecular Process Leading to Age-Related Loss of Function

Age-related metastatic mineralization of soft tissues has been considered a passive and spontaneous process. Recent data have demonstrated that calcium salt deposition in soft tissues could be a highly regulated process. Although calcification occurs in any tissue type, vascular calcification has been of particular interest due to association with atherosclerosis, chronic kidney disease (CKD), and osteoporosis. Different mechanisms underlying calcium apatite accumulation are explored with these age-related disorders. In the case of atherosclerotic plaques, oxy-lipids trigger release of the pro-inflammatory cytokines and inflammation that activate calcification processes in aorta intimae. In CKD patients, renal failure alters the balance between calcium and phosphate levels usually regulated by fibroblast growth factor-23 (FGF23), Klotho, and vitamin D, and vascular smooth muscle cells (VSMCs) begin to explore an osteoblastosteoblast-like phenotype. Calcification could affect extracellular matrix along with VSMCs. Collagen is a major component of extracellular matrix and its modifications accumulate with age. The formation of cross-links between collagen fibers is regulated by the action of lysine hydroxylases and lysyl oxidase and could occur spontaneously. Oxidation-induced advanced glycation end products (AGEs) are a major type of spontaneous cross-links that accelerate with age and may result in tissue stiffness, problems with recycling, and potential accumulation of calcium apatite. Applying strategies for clearing the AGEs proposed by de Grey may be more difficult in the highly mineralized extracellular matrix. We performed bioinformatic analysis of the molecular pathways underlying calcification in atherosclerotic and CKD patients, signaling pathways of collagen cross-links formation, and bone mineralization, and we propose new potential targets and review drugs for calcification treatment.

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.

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.

The Antioxidant Cofactor Alpha-Lipoic Acid May Control Endogenous Formaldehyde Metabolism in Mammals

The healthy human body contains small amounts of metabolic formaldehyde (FA) that mainly results from methanol oxidation by pectin methylesterase, which is active in a vegetable diet and in the gastrointestinal microbiome. With age, the ability to maintain a low level of FA decreases, which increases the risk of Alzheimers disease and dementia. It has been shown that 1,2-dithiolane-3-pentanoic acid or alpha lipoic acid (ALA), a naturally occurring dithiol and antioxidant cofactor of mitochondrial α-ketoacid dehydrogenases, increases glutathione (GSH) content and FA metabolism by mitochondrial aldehyde dehydrogenase 2 (ALDH2) thus manifests a therapeutic potential beyond its antioxidant property. We suggested that ALA can contribute to a decrease in the FA content of mammals by acting on ALDH2 expression. To test this assumption, we administered ALA in mice in order to examine the effect on FA metabolism and collected blood samples for the measurement of FA. Our data revealed that ALA efficiently eliminated FA in mice. Without affecting the specific activity of FA-metabolizing enzymes (ADH1, ALDH2, and ADH5), ALA increased the GSH content in the brain and up-regulated the expression of the FA-metabolizing ALDH2 gene in the brain, particularly in the hippocampus, but did not impact its expression in the liver in vivo or in rat liver isolated from the rest of the body. After ALA administration in mice and in accordance with the increased content of brain ALDH2 mRNA, we detected increased ALDH2 activity in brain homogenates. We hypothesized that the beneficial effects of ALA on patients with Alzheimers disease may be associated with accelerated ALDH2-mediated FA detoxification and clearance.

“Matreshka” genes with alternative reading frames

Although a relatively small part of the human genome contains protein encoding genes, the latest data on the discovery of alternative open reading frames (ORFs) in conventional mRNAs has highlighted the expanded coding potential of these genes. Until recently, it was believed that each mRNA transcript encodes a single protein. Recent proteogenomics data indicate the existence of exceptions to this rule, which greatly changes the usual meaning of the term “gene.” The topology of a gene with overlapping ORFs resembles a Russian “matreshka” toy. There are two levels of “matreshka” genetic systems. First, the chromosomal level, when the “nested” gene is located within introns and exons of the main chromosomal gene, both in the sense and antisense orientation relative to the external gene. The second level is a mature mRNA molecule containing overlapping ORFs or an ORF with an alternative start codon. In this review, we will focus on the properties of “matreshka” genes of the second type and methods for their detection and verification. Particular attention is paid to the biological properties of the polypeptides encoded by these genes.