Role of extracellular GAPDH in Streptococcus pyogenes virulence.

Pathogens generate molecules, or virulence factors, that enable them to colonize host tissues through several mechanisms, including adhesion to host tissues, or superior invasive capability. Some allow the pathogen to evade the host's immune system. Many of these molecules are proteins that are exported to the cell's surface or secreted. Curiously, GAPDH, which is a glycolytic enzyme, is also a virulence factor that has been shown to contribute to Streptococcus pyogenes pathogenicity by each of these mechanisms.

Basic biology of GAPDH.

The GAPDH gene is highly conserved with a promoter that contains several types of regulatory elements, perhaps even in a distal intron. Curiously, the transcription start site shows some ambiguity and there are codon-sharing exons at alternate exon junctions. While there is only one functional gene for GAPDH in humans, the genome is littered with pseudogenes, representing a trove of researchable content. Tissue-specific expression speaks to the glycolytic function of GAPDH; thus, it's not surprising to see expression increased in cancer cells. Modulation of protein levels becomes an opportunity for intervention. The abundance of GAPDH in the cell provides the rationale (albeit, tenuous) for its use as a loading control. The single paralogous GAPDHS, which is the spermatogenic form of the protein, provides a curious study in cell-type specificity and perhaps intervention (i.e. contraception). And it is no wonder that great biochemists were kept busy for decades unveiling the nuances of GAPDH enzymology. While the active site of the enzyme is well-characterized and the catalytic mechanism is well-described, the role of inter-subunit interactions in catalysis still offers some mysteries, particularly with regards to other emerging enzymatic properties. The GAPDH protein exhibits an intrinsic asymmetry of the subunits, which also may speak to its functional diversity.

GAPDH and intermediary metabolism.

GAPDH plays a major enzymatic role in the intermediary metabolism of human tissues. In fact, the cells of all organisms require the catalytic capability of GAPDH in order to maintain adequate glycolytic flux. Even the primitive archaea rely on GAPDH in a pivotal step in the Entner-Doudoroff pathway, which is a series of reactions that resembles glycolysis. GAPDH catalyzes the sixth reaction of glycolysis in eukaryotic cells and represents a regulatory hurdle in anaerobic glycolysis. The triose substrate of GAPDH is actually a product of several important metabolic pathways: stage one of glycolysis, fructose catabolism, pentose phosphate pathway and glycerol metabolism. The GAPDH reaction is reversible, hence, necessary for hepatic gluconeogenesis. The chapter discusses GAPDH as being a metabolic 'switching station', diverting carbon flow appropriately. There is discussion regarding the experimental analysis of GAPDH's enzymatic function, particularly in the use of inhibitors. The GAPDH gene is portrayed in the context of the enzyme's role in metabolism. The observed intolerance to genetic mutation suggests that the genetic changes (i.e. those seen across species) may provide a treasure of information regarding the limits of genetic variability that can be tolerated and still allow for the protein to conduct essential glycolytic - as well as non-glycolytic - functions.

Compartmentation of GAPDH.

The concept of the cytosol as a space that contains discrete zones of metabolites is discussed relative to the contribution of GAPDH. GAPDH is directed to very specific cell compartments. This chapter describes the utilization of GAPDH's enzymatic function for focal demands (i.e. ATP/ADP and NAD(+)/NADH), and offers a speculative role for GAPDH as perhaps moderating local concentrations of inorganic phosphate and hydrogen ions (i.e. co-substrate and co-product of the glycolytic reaction, respectively). Where known, the structural features of the binding between GAPDH and the compartment components are discussed. The nuances, which are associated with the intracellular distribution of GAPDH, appear to be specific to the cell-type, particularly with regards to the various plasma membrane proteins to which GAPDH binds. The chapter includes discussion on the curious observation of GAPDH being localized to the external surface of the plasma membrane in a human cell type. The default perspective has been that GAPDH localization is synonymous with compartmentation of glycolytic energy. The chapter discusses GAPDH translocation to the nucleus and to non-nuclear cellular structures, emphasizing its glycolytic function. Nevertheless, it is becoming clear that alternate functions of GAPDH play a role in compartmentation, particularly in the translocation to the nucleus.

Functional diversity.

There is increasing evidence to support a gene economy model that is fully based on the principles of evolution in which a limited number of proteins does not necessarily reflect a finite number of biochemical processes. The concept of 'gene sharing' proposes that a single protein can have alternate functions that are typically attributed to other proteins. GAPDH appears to play this role quite well in that it exhibits more than one function. GAPDH represents the prototype for this new paradigm of protein multi-functionality. The chapter discusses the diverse functions of GAPDH among three broad categories: cell structure, gene expression and signal transduction. Protein function is curiously re-specified given the cell's unique needs. GAPDH provides the cell with the means of linking metabolic activity to various cellular processes. While interpretations may often lead to GAPDH's role in meeting focal energy demands, this chapter discusses several other very distinct GAPDH functions (i.e. membrane fusogenic properties) that are quite different from its ability to catalyze oxidative phosphorylation of the triose, glyceraldehyde 3-phosphate. It is suggested that a single protein participates in multiple processes in the structural organization of the cell, controls the transmission of genetic information (i.e. GAPDH's involvement may not be finite) and mediates intracellular signaling.

GAPDH, as a virulence factor.

Pathogens, such as bacteria, viruses, protozoa and fungi, generate molecules that provide them with a selective advantage, often at the expense of the host. These molecules, or virulence factors, enable pathogens to colonize the host through several mechanisms. Some molecules offer the pathogen an advantage through better adhesion to host tissues, or superior invasive capability. Some allow the pathogen to evade or suppress the host's immune system. Some molecules enable intracellular parasites to disable cytoprotective mechansims, by re-directing the host phagocytic vesicles. Many of these molecules are proteins that are exported to the cell's surface or are secreted. As unlikely as it seems, GAPDH appears to play a role as a virulence factor in a number of pathogenic organisms by the mechanisms just described. This highly conserved protein is found on the outer surface or as a secretory product of these organisms. The process by which pathogenic GAPDH, which has >40 % sequence identity to human GAPDH, is exported and attached to the outer surface of cells remains unknown. This chapter also presents a previously unpublished proposed docking sequence on GAPDH. There is also discussion of the potential of using the antigenic properties of pathogenic GAPDH for medical as well as for veterinary purposes.

Target for diverse chemical modifications.

The chapter begins with an historical perspective of GAPDH isozymes that is juxtaposed to the fact that there is only one somatic functional gene in humans that is virtually identical among the mammalian species. Over the many years of GAPDH research, dozens of labs have reported the existence of multiple forms of GAPDH, which mostly vary as a function of charge with an occasional report of truncated forms. These observations are in part due to GAPDH being a substrate for many enzymatically-controlled post-translational modifications. While target residues have been identified and predictive algorithms have implicated certain residues, this area of research appears to be in its infancy regarding GAPDH. Equally fascinating, the uniquely susceptible nature of GAPDH to non-enzymatic reactions, that typically are associated with cell stress, such as oxidation and nitration, is also discussed. Two metabolic gases, nitric oxide and hydrogen sulfide, which are enzymatically produced, appear to exert their signaling properties through non-enzymatic reaction with GAPDH. Models of cellular decline are also proposed, including the compelling hypothesis that states cell compromise occurs by the physically blocking the function of chaperonins (i.e. dual-ring multiple-subunit molecular chaperones) by the attachment of misfolded GAPDH.

Multiple binding partners.

GAPDH interacts with a plethora of diverse cellular proteins. The network of interacting partners, or interactome, is presented for GAPDH with the interacting molecules grouped into specific functional and structural categories. By organizing the binding partners in this way, certain common structural features are beginning to surface, such as acidic dipeptide sequences that are found in several of these binding proteins. Additionally, the consensus sequences for target polynucleotides are being brought to light. The categories, which are presented according to function, offer an opportunity for research into the corresponding structural correlates to these interactions. Recent discoveries of interacting proteins have revealed novel relationships that are generating emerging mechanisms. Proteins that are associated with age-related neurodegenerative diseases appear to be particularly prone to binding GAPDH, suggesting that GAPDH may be playing a role in these diseases. Neurodegenerative diseases that are discussed are the conformational diseases of aging, suggesting that GAPDH may be a global sensor for cellular conformational stress. In addition to GAPDH's oxidoreductase activity, several other enzymatic functions have been discovered, including peroxidase, nitrosylase, mono-ADP-ribosylase and kinase activities.

Dynamic oligomeric properties.

This chapter provides a foundation for further research into the relationship between dynamic oligomeric properties and functional diversity. The structural basis that underlies the conformational sub-states of the GAPDH oligomer is discussed. The issue of protein stability is given a thorough analysis, since it is well-established that the primary strategy for protein oligomerization is to stabilize conformation. Several factors that affect oligomerization are described, including chemical modification by synthetic reagents. The effects of native substrates and coenzymes are also discussed. The curious feature of chloride ions having a de-stabilizing effect on native GAPDH structure is described. Additionally, the role of adenine dinucleotides in tetramer-dimer equilibrium dynamics is suggested to be a major part of the physiological regulation of GAPDH structure and function. This chapter also contends that a vast amount of useful information can come from comparative analyses of diverse species, particularly regarding protein stability and subunit-subunit interaction. Lastly, the concept of domain exchange is introduced as a means of understanding the stabilization of dynamic oligomers, suggesting that inter-subunit contacts may also be a way of masking docking sites to other proteins.

GAPDH in anesthesia.

Thus far, two independent laboratories have shown that inhaled anesthetics directly affect GAPDH structure and function. Additionally, it has been demonstrated that GAPDH normally regulates the function of GABA (type A) receptor. In light of these literature observations and some less direct findings, there is a discussion on the putative role of GAPDH in anesthesia. The binding site of inhaled anesthetics is described from literature reports on model proteins, such as human serum albumin and apoferritin. In addition to the expected hydrophobic residues that occupy the binding cavity, there are hydrophilic residues at or in very close proximity to the site of anesthetic binding. A putative binding site in the bacterial analog of the human GABA (type A) receptor is also described. Additionally, GAPDH may also play a role in anesthetic preconditioning, a phenomenon that confers protection of cells and tissues to future challenges by noxious stimuli. The central thesis regarding this paradigm is that inhaled anesthetics evoke an intra-molecular protein dehydration that is recognized by the cell, eliciting a very specific burst of chaperone gene expression. The chaperones that are implicated are associated with conferring protection against dehydration-induced protein aggregation.

Protection against protein aggregation by alpha-crystallin as a mechanism of preconditioning.

Anesthetic preconditioning occurs when cells previously exposed to inhaled anesthetics are protected against subsequent injury. We hypothesize that inhaled anesthetics may cause slight protein misfolding that involves site-specific dehydration, stimulating cytoprotective mechanisms. Human neuroblastoma cells were exposed to ethanol (as the dehydration agent) followed by quantitative analysis of the expression of five heat shock genes: DNAJC5G, CRYAA, HSPB2, HSF4 and HSF2. There was an ethanol-induced upregulation of all genes except HSF4, similar to previous observations using isoflurane. CRYAA (the gene for alphaA-crystallin) exhibited a 23.19 and 17.15-fold increase at 24 and 48 h post ethanol exposure, respectively. Additionally, we exposed glyceraldehyde 3-phosphate dehydrogenase to ethanol, which altered oligomeric subspecies and caused protein aggregation in a concentration-dependent manner. Ethanol-mediated dehydration-induced protein aggregation was prevented by incubation with alpha-crystallin. These data indicate that ethanol mimics the effects of isoflurane presumably through a cellular preconditioning mechanism that involves dehydration-induced protein aggregation.

Isoflurane's Effect on Protein Conformation as a Proposed Mechanism for Preconditioning.

Persistent alteration of protein conformation due to interaction with isoflurane may be a novel molecular aspect of preconditioning. We preincubated human serum albumin with isoflurane, dialyzed to release agent, and assessed protein conformation. Susceptibility to chemical modification by methylglyoxal and nitrophenylacetate was also examined. Isoflurane had a persistent effect on protein conformation. An increase in the susceptibility of surface residues to chemical modification attended this change in conformation. Modification of isoflurane-treated HSA included intra- and intersubunit cross-linking that may be a consequence of anesthetic-induced changes in multimeric subpopulations. This irreversible effect of isoflurane may represent a mechanism for preconditioning.

Isoflurane preconditioning involves upregulation of molecular chaperone genes.

Isoflurane preconditioning is a phenomenon in which cells previously exposed to isoflurane exhibit protection against subsequent noxious stimuli. We hypothesize that isoflurane may cause subtle protein misfolding that persists at a sublethal level, stimulating cytoprotective mechanisms. Human neuroblastoma cells (SH-SY5Y) were exposed to isoflurane followed by quantitative analysis of the expression of several families of heat shock genes (84 total transcripts). Our data is consistent with a model of an early and delayed phase of preconditioning. Different patterns of expression of the 84 genes were seen at 1 and 24h post-isoflurane exposure. Expression of 45 of the 84 genes were elevated at 1h (or early phase) and remained upregulated at 24h (or delayed phase). Subsets of the remaining genes were either unchanged (13 genes), early-specific upregulated (17 genes) or delayed-specific upregulated (9 genes). We also demonstrated that isoflurane caused a slight yet detectable misfold of a model protein. These data indicate that brief anesthetic exposure promotes specific patterns of gene expression, leading to preconditioning which would enhance the cell's ability to tolerate a future injury that involves protein misfolding.

Carnosine protects against the neurotoxic effects of a serotonin-derived melanoid.

Anesthesia-related postoperative cognitive dysfunction (POCD) leads to morbidity in the elderly. Lipid peroxidative byproducts (i.e. acrolein) accumulate in aging and may play a role. Sevoflurane, an inhaled anesthetic, sequesters acrolein and enhances the formation of a serotonin-derived melanoid (SDM). SDM may be a biologically relevant polymeric melanoid that we previously showed exhibits redox activity and disrupts lipid bilayers. In this study, we examined the toxicity of SDM in cell culture and looked at protection using L-carnosine. SDM's toxic effects were tested on neuronal-like SH-SY5Y cells, causing an exponential decrease in viability, while human dermal fibroblasts were completely resistant to the toxic effects. SDM brought about morphological changes to differentiated SH-SY5Y cells, particularly to neuronal processes. Co- but not pre-treatment with L-carnosine protected differentiated SH-SY5Y cells exposed to SDM. Our mechanism suggests focal sevoflurane-induced sequestration of age-related acrolein leading to SDM synthesis and neuronal impairment, which is prevented by L-carnosine.

Redox mechanism of neurotoxicity by a serotonin-acrolein polymeric melanoid.

Postoperative cognitive dysfunction may be associated with the toxic products of lipid peroxidation, such as the α,ß-unsaturated aldehyde acrolein, which accumulates in aging. We previously identified an acrolein-mediated, serotonin-derived melanoid product, or SDM. This study further characterizes this putative novel neuromelanin, which is not made from catecholamines. In addition to its strong protein-binding properties, we observed that SDM binds Fe(2+) readily and exhibits complex redox characteristics. SDM may exist as a two-dimensional network of polymers that coalesce into larger entities exhibiting electroactive properties. These observations suggest that SDM may contribute to the decline in cognition due to focal degeneration from SDM-mediated free-radical production. We know that inhalational anesthetics sequester acrolein, which is toxic to neurons, and we propose that the local increase in acrolein depletes serotonin levels and enhances neuronal vulnerability through the production of neuromelanin-like structures, such as SDM.

Inhaled Anesthetics Promote Albumin Dimerization through Reciprocal Exchange of Subdomains.

Inhaled anesthetics affect protein-protein interaction, but the mechanisms underlying these effects are still poorly understood. We examined the impact of sevoflurane and isoflurane on the dimerization of human serum albumin (HSA), a protein with anesthetic binding sites that are well characterized. Intrinsic fluorescence emission was analyzed for spectral shifting and self-quenching, and control first derivatives (spectral responses to changes in HSA concentration) were compared against those obtained from samples treated with sevoflurane or isoflurane. Sevoflurane increased dimer-dependent self-quenching and both decreased oligomer-dependent spectral shifting, suggesting that inhaled anesthetics promoted HSA dimerization. Size exclusion chromatography and polarization data were consistent with these observations. The data support the proposed model of a reciprocal exchange of subdomains to form an HSA dimer. The open-ended exchange of subdomains, which we propose occuring in HSA oligomers, was inhibited by sevoflurane and isoflurane.

Isoflurane's effect on interfacial dynamics in GAPDH influences methylglyoxal reactivity.

Diabetic surgical patients are at risk for peri- and post-operative complications, which can be prevented by maintaining tight glycemic control during anesthesia. Control of blood sugar would decrease unwanted chemical reactions, such as protein glycation, minimizing tissue dysfunction. Methylglyoxal (MG) is a major contributor to protein modification and tissue dysfunction seen in diabetic patients. We hypothesized that inhaled anesthetics may play a role in protein glycation and examined the effects of isoflurane on MG-induced modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Isoflurane promoted MG-induced modification of GAPDH as evidenced by an increase in fluorescent glycation products, a change in chromatographic elution patterns and a loss of enzyme activity. Isoflurane's effect may be mediated by altering interfacial events. Our working model involves the binding of isoflurane to GAPDH, increasing the susceptibility to MG-induced modification of residues involved in oligomerization. These findings suggest a molecular basis for maintaining glycemic control during anesthesia.

Toxicity of a serotonin-derived neuromelanin.

Postoperative Cognitive Dysfunction (POCD) is associated with increased mortality in the elderly and may occur from lipid peroxidation in aging. We previously showed that sevoflurane sequesters acrolein, which promotes the formation of a novel species of a putative neuromelanin. The current study examined the properties of this serotonin-derived melanoid (SDM). The interaction of SDM with unilamellar vesicles (ULVs) was examined using lipid membrane probes. Vesicle disruption was investigated by leakage of dye from calcein-loaded ULVs. We observed that SDM decreased diphenyl-hexatriene fluorescence anisotropy and increased the temperature-dependent change in anisotropy. SDM changed the absorbance of merocyanin-bound ULVs. SDM increased detergent-mediated calcein leakage. SDM structure was dramatically altered upon interaction with ULVs. We also observed that SDM enhanced detergent-mediated leakage of loaded ULVs, suggesting that SDM may be neurotoxic. We propose that inhalational agents, which sequester acrolein, may promote the production of certain species of neuromelanin that depletes local serotonin and enhances neuronal vulnerability.

Interfacial effects on the conformation of amyloid-beta peptide.

We examined the effects of air-water and water-sevoflurane interfaces on conformational properties of amyloid-beta peptide (ABP). Fractions were extracted from sub-interfacial (air-water) and supra-interfacial (water-sevoflurane) layers and compared with aqueous bulk layers using fluorescence properties of ABP provided by a single tyrosine. The observations suggest that interfacial ABP may be more disordered than bulk ABP.

Trifluoroethanol increases albumin's susceptibility to chemical modification.

Acrolein-dependent chemical modification is implicated in the etiology of postoperative cognitive dysfunction (POCD). We examined this process further using human serum albumin (HSA), which is a target of acrolein modification and contains anesthetic binding sites. We tested whether trifluoroethanol (TFE), which mimics inhaled anesthetics, affects the susceptibility of HSA to modification by acrolein. We observed that acrolein promoted the formation of fluorescent adducts. TFE (10%) increased the amount of acrolein-HSA adducts. TFE (40%) caused a 5-fold increase in adduct formation. Acrolein also increased tryptophan anisotropy of HSA, which was further increased by TFE (10%). Acrolein-induced protein cross-linking was also increased in the presence of TFE (40%). These observations suggest that TFE promotes acrolein-induced modification of HSA, supporting a putative mechanism for POCD.