Transglutaminases - possible drug targets in human diseases

Transglutaminases (TGases) belong to a family of closely related proteins that catalyze the cross linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate with the formation of an Nepsilon-(gamma-L-glutamyl)-L-lysine [GGEL] cross link and the concomitant release of ammonia. Such cross-linked proteins are often highly insoluble. Neurodegenerative diseases, such as Alzheimer disease (AD), Parkinson disease (PD), supranuclear palsy and Huntington disease (HD), are characterized in part by aberrant cerebral TGase activity and by increased cross-linked proteins in affected brain. In support of the hypothesis that TGases contribute to neurodegenerative disease, a recent study shows that knocking out TGase 2 in HD-transgenic mice results in increased lifespan. Moreover, recent studies show that cystamine, an in vitro TGase inhibitor, prolongs the lives of HD-transgenic mice. However, these findings are not definitive proof of TGase involvement in HD neuropathology. In neurodegenerative diseases, the brain is under oxidative stress and cystamine can theoretically be converted to the potent antioxidant cysteamine in vivo. Cystamine is also a caspase 3 inhibitor. In addition to neurodegenerative diseases, aberrant TGase activity is associated with celiac disease. Interestingly, a subset of celiac patients develops neurological disorders. This review focuses on the strategies that have been recently employed in the design of TGase inhibitors, and on the possible therapeutic benefits of selective TGase inhibitors to patients with neurodegenerative disorders or to patients with celiac disease.     

Transglutaminases as targets for pharmacological inhibition

Transglutaminases (TGases), a family of enzymes that catalyze the formation of epsilon-(gamma-glutamyl)lysine isopeptide linkage, play an important physiological role in hemostasis, wound healing, assembly and remodeling of the extracellular matrix, cell signaling and apoptosis. Although many members of this class of enzymes have been known for decades, their role in various physiological and pathological processes is still a subject of substantial research and debate. Convincing evidence exists that TGases are involved in formation of cytotoxic proteinatious aggregates in Alzheimer's, Huntington's and other neurodegenerative diseases. However, it is not clear if elevated levels of TGases play a causative or protective role in several of these processes. Increased or defective TGase activity is a factor in cortical cataract formation, lamellar ichtyosis and fibrosis. TGase creates epitopes for the production of autoantibodies in celiac disease and possibly other autoimmune diseases. Another TGase, Factor XIIIa, is involved in the etiology of vascular diseases. Modulation of TGase activity through its selective inhibition may have therapeutic benefit in a wide variety of diseases. This paper will examine TGases as targets for the development of new therapeutics and review the progress in discovery of selective inhibitors of these enzymes.     

Role of transglutaminase-catalyzed reactions in the post-translational modifications of proteins responsible for immunological disorders

Transglutaminases (TG, E.C. 2.3.2.13) are a family of related and ubiquitous enzymes which catalyze the cross linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. These enzymes are also capable of catalyzing other reactions which are important for cell life. The distribution and the physiological roles of human TGs have been widely studied in numerous cell types and tissues and recently their roles in several diseases have begun to be identified. It has been hypothesized that transglutaminase activity is directly involved in the pathogenetic mechanisms responsible for several human diseases. In particular, "tissue" TG (tTG, type 2), a member of the TG enzyme family, has been recently shown to be involved in the molecular mechanisms responsible for a very widespread human pathology, Celiac Disease (CD), which is characterized, in part, by aberrant transglutaminase activity and by the presence of transglutaminase-modified proteins. In this review we describe the biochemistry of TGs, with particular reference to the molecular mechanisms involved in the physiopathology of this human disease, as a model for the study of other immunological disorders.     

Pathophysiological roles of transglutaminase - catalyzed reactions in the pathogenesis of human diseases

Transglutaminases (TGs, E.C. 2.3.2.13) are related and ubiquitous enzymes which catalyze the cross linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. These enzymes are also capable of catalyzing other reactions which are important for cell life. To date, at least eight different human TGs have been identified. The distribution and the physiological roles of human TGs have been widely studied in numerous cell types and tissues and recently their roles in several diseases have begun to be identified. It has been hypothesized that transglutaminase activity is directly involved in the patho-genetic mechanisms responsible for several human diseases. In particular, TG2, a member of the TG enzyme family, has been shown to be involved in the molecular mechanisms responsible for a very widespread human pathology, Celiac Disease (CD), one of the most common food intolerances described in the western population. The main food agent that provokes the strong and diffuse clinical symptoms has been known for several years to be gliadin, a protein present in a very large number of human foods derived from vegetables. The aim of this review is to summarize the most recent findings concerning the relationships between the biochemical properties of the transglutaminase activity and the basic molecular mechanisms responsible for CD. In addition, we present some clinical associations of CD with other human diseases, with particular reference to neuropsychiatric disorders. Possible molecular links between biochemical activities of transglutaminase enzymes, CD and neuropsychiatric disorders are discussed.     

Assay of transglutaminase activity by electrophoretic removal of the unreacted monodansyl cadaverine

Transglutaminases (TGases) catalyze the transfer of acyl groups between the γ-carboxyamide group of a glutamine residue and a primary amine. Rapid and precise determination of TGase activity is an important issue because improper function of TGases has been suggested to be associated with a variety of diseases. There have been tremendous efforts to develop the TGase assay methods to be more rapid, convenient and accurate. In the conventional assay method, fluorescence-tagged amine molecules such as monodansyl cadaverine (MDC) are coupled with casein by the action of transglutaminase. The removal step of unreacted MDC would require time-consuming work-up processes such as acid-precipitation and centrifugation. These processes would also interrupt the precise measurements of enzymatic activities. In this study, we have developed a new fluorometric assay methods to assay transglutaminase activity based on electrodialysis where the unreacted MDC is removed by electrophoresis. We have found the optimized condition to remove the unreacted MDC while preserving the β-casein protein. We also found the linear relationship between fluorescence intensity associated with β-casein and TGase can maintain in the range of 0–1.6 mU as well as 0–0.4 mU. The results show us as few as 0.1 mU of TGase could be detected by this method.     

Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase

Transglutaminase (TGase, E.C. 2.3.2.13) catalyzes acyl transfer reactions between the gamma-carboxamide groups of protein-bound glutamine (Gln) residues, which serve as acyl donors, and primary amines, resulting in the formation of new gamma-amides of glutamic acid and ammonia. By using an amino-derivative of poly(ethylene glycol) (PEG-NH(2)) as substrate for the enzymatic reaction with TGase it is possible to covalently bind the PEG polymer to proteins of pharmaceutical interest. In our laboratory, we have conducted experiments aimed to modify proteins of known structure using TGase and, surprisingly, we were able to obtain site-specific modification or PEGylation of protein-bound Gln residue(s) in the protein substrates. For example, in apomyoglobin (apoMb, myoglobin devoid of heme) only Gln91 was modified and in human growth hormone only Gln40 and Gln141, despite these proteins having many more Gln residues. Moreover, we noticed that these proteins suffered highly selective limited proteolysis phenomena at the same chain regions being attacked by TGase. We have analysed also the results of other published experiments of TGase-mediated modification or PEGylation of several proteins in terms of protein structure and dynamics, among them alpha-lactalbumin and interleukin-2, as well as disordered proteins. A noteworthy correlation was observed between chain regions of high temperature factor (B-factor) determined crystallographically and sites of TGase attack and limited proteolysis, thus emphasizing the role of chain mobility or local unfolding in dictating site-specific enzymatic modification. We propose that enhanced chain flexibility favors limited enzymatic reactions on polypeptide substrates by TGases and proteases, as well as by other enzymes involved in a number of site-specific post-translational modifications of proteins, such as phosphorylation and glycosylation. Therefore, it is possible to predict the site(s) of TGase-mediated modification and PEGylation of a therapeutic protein on the basis of its structure and dynamics and, consequently, the likely effects of modifications on the functional properties of the protein.     

Transglutaminase inhibitors: a patent review

Introduction: Transglutaminases (TGases) are a class of enzymes that play multifunctional roles. Their protein-crosslinking activity has been linked to fibrosis and Huntington’s disease, their glutamine deamidation activity has been related to celiac disease and their GTP-binding activity has been implicated in cancer. All of these physiological disorders have prompted the development of inhibitors, which has accelerated dramatically over the past decade.

Areas covered: This review presents an overview of TGase inhibitors published in the patent literature, from the first compounds developed in the late 1980’s, to the current date. This article is focussed on the chemical structure of new inhibitors and their probable mechanism of action.

Expert opinion: Comparison of effective TGase inhibitors reveals common structural features that may guide future design. Many of these elements are embodied in the first TGase inhibitor to recently enter into clinical trials.     

Enzymatic procedure for site-specific pegylation of proteins

We have developed a novel methodology for site-specific pegylation of proteins by use of transglutaminase (TGase). In this methodology, alkylamine derivatives of poly(ethyleneglycol) (PEG) could be site-specifically incorporated into intact or chimeric proteins without decreasing the bioactivities. The incorporation site of the TGase-catalyzed modification is limited to the substrate Gln residues for TGases. The high homogeneity of the constructed conjugates and the ability to design conjugates with suitable incorporation sites will improve the applicability of PEG-protein conjugates for clinical use.     

Roles of plasma proteins in the formation of silicotic nodules in rats

The contribution of plasma protein(s) to the stabilization of fibroids formed in rat lungs exposed to acute silica dust inhalation was examined. Antibodies against component proteins of the nodules remaining insoluble in 2% SDS, 10M urea and 40 mM sulfhydryl reagents under prolonged boiling conditions were raised in rabbits and used to capture plasma proteins, which were identified by 2D-gel electrophoresis and MALDI-TOF analysis. The silica particles were encapsulated with extracellular protein composites whose amino acid compositions showed high levels of alanine, i.e., above those of glycine and proline, a building block of collagen. Antibody-captured plasma proteins showed the dominant presences of fibrinogen, albumin, and prealbumin (transthyretin), and other minor proteins, which included alpha-1-protease inhibitor, contraspin-like protease inhibitor, cathepsin B, etc. The presence of the N(epsilon)-(gamma-glutamyl) lysine isopeptide bond in the nodules was evidenced by direct chemical methods and by immunoreactivity for anti-isopeptide bonds. Immunostaining of affected lung tissue and of the fibroid regions showed elevated levels of transglutaminase (TGase) E and plasma factor XIII (F-XIII), but showed no reactivity towards other TGases. These findings suggest that the silica encapsulated nodules are a mixture of extracellular proteins that include collagen type I, fibrin and transthyretin, which is stabilized by TGase catalyzed crosslinking between plasma and extracellular proteins during fibrosis to eventually form insoluble nodules.     

Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase

Abstract: Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln¹⁶, which was identified as the amino acceptor substrate. Different γ(glutamyl¹⁶)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono-dansyl-cadaverine of the peptide were purified by high-performance liquid chromatography on a reverse-phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys²¹, was demonstrated to be involved in both inter- and intramolecular cross-link formation.     

Tissue transglutaminase: a novel pharmacological target in preventing toxic protein aggregation in neurodegenerative diseases

Alzheimer's disease, Parkinson's disease and Huntington's disease are neurodegenerative diseases, characterized by the accumulation and deposition of neurotoxic protein aggregates. The capacity of specific proteins to self-interact and form neurotoxic aggregates seems to be a common underlying mechanism leading to pathology in these neurodegenerative diseases. This process might be initiated and/or accelerated by proteins that interact with these aggregating proteins. The transglutaminase (TG) family of proteins are calcium-dependent enzymes that catalyze the formation of covalent ε-(γ-glutamyl)lysine isopeptide bonds, which can result in both intra- and intermolecular cross-links. Intramolecular cross-links might modify self-interacting proteins, and make them more prone to aggregate. In addition, intermolecular cross-links could link self-aggregating proteins and thereby initiate and/or stimulate the aggregation process. So far, increased levels and activity of tissue transglutaminase (tTG), the best characterized member of the TG family, have been observed in many neurodegenerative diseases, and the self-interacting proteins, characteristic of Alzheimer's disease, Parkinson's disease and Huntington's disease, are known substrates of tTG. Here, we focus on the role of tTG in the initiation of the aggregation process of self-interacting proteins in these diseases, and promote the notion that tTG might be an attractive novel target for treatment of neurodegenerative diseases.     

Amine oxidases of the quinoproteins family: their implication in the metabolic oxidation of xenobiotics

Copper amine oxidases (CuAOs) are ubiquitous enzymes, which play a vital role in the physiology and pathology of mammals in controlling the metabolism of various primary monoamines, diamines and polyamines of endogenous or xenobiotic origin. CuAOs, which belong to the quinoproteins family, possess two cofactors: tightly bound Cu^II and a quinone residue, which catalyzes the oxidative deamination of primary amines with concomitant production of aldehyde, ammonia and hydrogen peroxide through a “ping-pong” mechanism. Interest in human enzymes of the CuAOs class has increased in recent years driven by the discovery that the human vascular adhesion protein-1 (VAP-1), which regulates leucocyte trafficking and glucose transport, is a CuAO enzyme. The activities of CuAOs are increased in various human disorders such as diabetes, Alzheimer's disease and many inflammation-associated diseases leading to the overproduction of toxic metabolites, especially hydrogen peroxide and aldehyde compounds. As most consequences are pathological, effective and selective inhibitors of CuAOs should be of great interest as therapeutic agents. Nevertheless, the utilization of CuAOs to generate enzymatic toxic products into cancer cells for selective in situ killing, deserves to be considered in cancer therapy. This paper briefly highlights recent progress in the study of physiological, pathological and molecular aspects of CuAOs in mammals. Furthermore, a small molecule, that mimics the metabolic activity of CuAOs toward endogenous and exogenous amines, is described because it could be used as a surrogate of enzymes for a preliminary screening of potential inhibitors of CuAO enzymes.     

Functional Genomics- and Network-driven Systems Biology Approaches for Pharmacogenomics and Toxicogenomics

Recent advances in genomic technologies have enabled the identification of thousands of genetic variations that are associated with hundreds of complex human diseases or traits in genome-wide association studies (GWAS). The large number of genetic loci uncovered for each disease or trait along with the difficulty in pinpointing the underlying genes and mechanisms further testify to the complexity of human pathophysiology. To alleviate the challenges of GWAS, systems biology approaches have been utilized to map the molecular mechanisms underlying complex human diseases/traits via the integration of genetic variation, functional genomics (such as genetics of gene expression), pathways, and molecular networks. Similar approaches have been applied to a spectrum of drug metabolizing enzymes to discover novel functional genetic variations that affect the expression or activities of these enzymes as well as to define the regulatory pathways/networks of genes involved in drug metabolism and toxicology in key human tissues. We envision that the increased coverage of functional genetic polymorphisms, the availability of drug metabolism-centered gene networks, and the maturing methodologies previously developed for understanding complex human diseases can be applied to pharmacogenomic and toxicogenomic studies to further our understanding of inter-individual variability in drug efficacy and toxicity and eventually help direct personalized medicine.     

Molecular basis of human MAO A and B

Monoamine oxidase A and B (MAO A and B) catalyze the oxidative deamination of a number of biogenic and xenobiotic amines with different substrate and inhibitor specificities. Recently, cDNA clones that encode the human liver MAO A and B have been isolated. Comparison of the deduced amino acid sequences shows that they are 70% homologous and they appear to be derived from separate genes. Expression of functional enzymes by transient transfection of the cDNAs provide unequivocal evidence that the different catalytic activities of MAO A and B reside in their primary amino acid sequences. These two genes located on the X chromosome, Xp11.23, are deleted in some patients with Norrie disease. The possible role or the linkage of MAO genes to a number of diseases can now be investigated.     

The principle and the potential approach to ROS-dependent cytotoxicity by non-pharmaceutical therapies: optimal use of medical gases with antioxidant properties

Regulation of cellular redox balances is important for the homeostasis of human health. Thus, many important human diseases, such as inflammation, diabetes, glaucoma, cancers, ischemia and neurodegenerative diseases, have been investigated in the field of reactive oxygen species (ROS) and oxidative stress. To overcome the harmful effect of oxidative stress and ROS, one can directly eliminate them by medical gases such as carbon monoxide (CO), hydrogen sulphide (H(2)S), and molecular hydrogen (H(2)), or one can induce ROS-resistant proteins and antioxidant enzymes to antagonize oxidative stresses. This article reviews the molecular mechanisms how these medical gasses work as antioxidants, and how ROS resistant proteins are produced in the physiological context. Targeted therapeutic modalities to scavenge or prevent ROS might be applied in the prevention and treatment of ROS-related diseases in the near future.     

Pharmacological targeting of catalyzed protein folding: the example of peptide bond cis/trans isomerases

Peptide bond isomerases are involved in important physiological processes that can be targeted in order to treat neurodegenerative disease, cancer, diseases of the immune system, allergies, and many others. The folding helper enzyme class of Peptidyl-Prolyl-cis/trans Isomerases (PPIases) contains the three enzyme families of cyclophilins (Cyps), FK506 binding proteins (FKBPs), and parvulins (Pars). Although they are structurally unrelated, all PPIases catalyze the cis/trans isomerization of the peptide bond preceding the proline in a polypeptide chain. This process not only plays an important role in de novo protein folding, but also in isomerization of native proteins. The native state isomerization plays a role in physiological processes by influencing receptor ligand recognition or isomer-specific enzyme reaction or by regulating protein function by catalyzing the switch between native isomers differing in their activity, e.g., ion channel regulation. Therefore elucidating PPIase involvement in physiological processes and development of specific inhibitors will be a suitable attempt to design therapies for fatal and deadly diseases.     

Hormetic modulation of aging and longevity by mild heat stress

Aging is characterized by a stochastic accumulation of molecular damage, progressive failure of maintenance and repair, and consequent onset of age-related diseases. Applying hormesis in aging research and therapy is based on the principle of stimulation of maintenance and repair pathways by repeated exposure to mild stress. In a series of experimental studies we have shown that repetitive mild heat stress has anti-aging hormetic effects on growth and various other cellular and biochemical characteristics of human skin fibroblasts undergoing aging in vitro. These effects include the maintenance of stress protein profiles, reduction in the accumulation of oxidatively and glycoxidatively damaged proteins, stimulation of the proteasomal activities for the degradation of abnormal proteins, improved cellular resistance to ethanol, hydrogen peroxide and ultraviolet-B rays, and enhanced levels of various antioxidant enzymes. Anti-aging hormetic effects of mild heat shock appear to be facilitated by reducing protein damage and protein aggregation by activating internal antioxidant, repair and degradation processes.     

Hemorrhagic, fibrinogenolytic and edema-forming activities of the venom of the colubrid snake Philodryas olfersii (green snake).

The venom of P. olfersii has high hemorrhagic, edema-inducing and fibrin(ogen)olytic activities. It is devoid of thrombin-like, procoagulant, phospholipase A2 and platelet aggregating enzymes. The main activities are metalloproteinases inhibited by metal chelators (EDTA and 1,10-phenanthroline) and sulfhydryl compounds (DTT and cysteine). The hemorrhagic and fibrinogenolytic enzymes were partially purified by gel filtration on HPLC. The hemorrhagic activity of the venom was neutralized by commercial horse antivenoms to Bothrops species, as well as by rabbit antisera specific for hemorrhagic factors isolated from these Bothrops venoms. No immunoprecipitin reactions were obtained, indicating that the few epitopes of the P. olfersii hemorrhagin are involved in these neutralization reactions. The fibrinogenolytic enzyme cleaves A alpha-chain more quickly than the B beta-chain of human fibrinogen. The venom also solubilizes fibrin. This solubilization appears to occur from the hydrolysis of unpolymerized alpha-chain and cross-linked gamma-gamma dimer. The fibrin peptide products are distinct from those produced by plasmin.     

Enzymatically activatable diagnostic probes

Molecular imaging of disease development, progression and treatment is seen as key to further advancement in the understanding and triumph over illness. The role of enzymes is to catalyze the biochemical reactions that help regulate health, and when dysregulated in complex organisms lead to or indicate disease. The ability to image the action of these proteins for diagnostic purposes opens a window for the researcher and clinician to witness specifc molecular events in vitro and in vivo. Such probes have been developed and deployed for the optical, radionuclide and magnetic resonance modalities and offer significant benefits over conventional agents. The signal of enzymatically-activated probes is regulated by the specific activity of the desired enzyme. This allows for a higher signal to background ratio over non-specific and targeted agents. It also enables the modulation of contrast agent distribution (and even cellular accumulation) following enzymatic activity. This review summarizes the strategies and probes in development and use in this emergent field of molecular imaging, with a particular focus on the research and medical relevance of these advances.     

Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from Cucurbitaceae plants.

1. The biochemical characteristics and biological activities of eight Cucurbitaceae plant proteins designated trichosanthin (isolated from tubers of Trichosanthes kirilowii), beta-trichosanthin (isolated from tubers of Trichosanthes cucumeroides), alpha- and beta-momorcharins (isolated from seeds of Momordica charantia), momorchochin (isolated from tubers of Momordica cochinchinensis), luffaculin (isolated from seeds of Luffa acutangula) and luffin-a and luffin-b (isolated from seeds of Luffa cylindrica), were reviewed. 2. The isolation procedures for all eight proteins are based on aqueous extraction, acetone fractionation and ion exchange chromatography. Ammonium sulfate precipitation and gel filtration are steps which may be included to improve purification. 3. The proteins are basic in nature and possess a molecular weight of approx. 30,000. All except trichosanthin are glycoproteins. The content of Asx and Glx residues is high. The N-terminal amino acid residue is Asp. Their amino acid compositions and N-terminal amino acid sequences are similar. 4. Circular dichroism spectroscopic studies revealed that trichosanthin, alpha- and beta-momorcharins possess similar secondary but different tertiary structures. 5. Most of the proteins are immunologically distinct. 6. The proteins exhibit abortifacient, antitumor, ribosome inactivating and immunomodulatory activities. Trichosanthin manifests anti-human immunodeficiency virus activity.