Transglutaminase-catalyzed reactions responsible for the pathogenesis of celiac disease and neurodegenerative diseases: from basic biochemistry to clinic

Transglutaminases (TGases) are 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 with the formation of an N-gamma-(epsilon-L-glutamyl)-L-lysine [GGEL] cross link (isopeptidic bond) and the concomitant release of ammonia. Such cross-linked proteins are often highly insoluble. The TGases are closely related enzymes and can also catalyze other important reactions for cell life. Recently, several findings concerning the relationships between the biochemical activities of the TGases and the basic molecular mechanisms responsible for some human diseases, have been reported. For example, some neurodegenerative diseases, such as Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), supranuclear palsy, etc., are characterized in part by aberrant cerebral TGase activity and by increased cross-linked proteins in affected brains. Our article describes the biochemistry and the physio-pathological roles of the TGase enzymes, with particular reference to human pathologies in which the molecular mechanism of disease can be due to biochemical activities of the tissue TGase enzyme (tTGase, type 2), such as in a very common human disease, Celiac Disease (CD), and also in certain neuropsychiatric disorders.     

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.     

Potential of transglutaminase 2 as a therapeutic target

Importance of the field: Increased expression and activity of transglutaminase 2 – a calcium-dependent enzyme which catalyzes protein cross-linking, polyamination or deamidation at selective glutamine residues – are involved in the etiopathogenesis of several pathological conditions, such as neurodegenerative disorders, autoimmune diseases and inflammatory diseases. Inhibition of enzyme activity has potential for therapeutic management of these diseases.

Areas covered in this review: The major results achieved in the last twelve years of research in the field of inhibition of tranglutaminase activity using cell cultures as well as in vivo models of high-social-impact or widespread diseases, such as CNS neurodegenerative disorders, celiac sprue, cancer and fibrotic diseases.

What the reader will gain: Beneficial effects of enzyme activity inhibition have been observed in neurodegeneration and fibrosis in vivo models by delivery of the competitive inhibitor cystamine and more recently designed inhibitors, such as thiomidaziolium or norleucine derivatives, which irreversibly bind the active site cysteine residue. Transglutaminase 2 targeting with specific antibodies has also been shown to be a promising tool for celiac disease treatment.

Take home message: New insights from transglutaminase inhibition studies dealing with side effects of in vivo administration of pan-transglutaminase inhibitors will help in design of novel therapeutic approaches to various diseases.     

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.     

Targeting aggregation in the development of therapeutics for the treatment of Huntington's disease and other polyglutamine repeat diseases

Huntington's disease (HD) is one of a number of familial polyglutamine (polyQ) repeat diseases. These neurodegenerative disorders are caused by expression of otherwise unrelated proteins that contain an expansion of a polyQ tract, rendering them toxic to specific subsets of vulnerable neurons. These expanded repeats have an inherent propensity to aggregate; insoluble neuronal nuclear and cytoplasmic polyQ aggregates or inclusions are hallmarks of the disorders [1,2]. In HD, inclusions in diseased brains often precede onset of symptoms, and have been proposed to be involved in pathogenicity [3-5]. Various strategies to block the process of aggregation have been developed in an effort to create drugs that decrease neurotoxicity. A discussion of the effect of antibodies, caspase inhibitors, chemical inhibitors, heat-shock proteins, suppressor peptides and transglutaminase inhibitors upon aggregation and disease is presented.     

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.     

Apoptotic mechanisms involved in neurodegenerative diseases: experimental and therapeutic approaches

Alzheimer's disease (AD) and Parkinson's disease (PD) are two of the most significant neurodegenerative disorders in the developed world. However, although these diseases were described almost a century ago, the molecular mechanisms that lead to the neuronal cell death associated with these diseases are not yet clear, and vigorous research efforts have failed to identify effective treatment options. In the present review, we evaluate the potential mechanisms underlying apoptosis and neuronal death in neurodegenerative disorders. A role for mitochondria in the release of proapoptotic proteins, such as cytochrome c and apoptosis-inducing factor (AIF) etc., is discussed along with key processes involving oxidative stress and activation of glutamate receptors. We also deliberate the implication of DNA damage, primarily p53 induction and reentry in the cell cycle. Finally, we postulate that multitargeting therapies comprising antioxidants, cell cycle inhibitors and modulating agents of COX-2 or c-JUN kinase pathways could be suitable strategies to prevent or delay the process of neuronal cell death in neurodegenerative disorders. Thus, the aim of this review is to discuss the pathways involved in the pathogenesis of neurodegenerative diseases such as AD, PD and Huntington's disease (HD). Furthermore, current and future pharmacotherapeutics will be considered.     

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.     

Removing protein aggregates: the role of proteolysis in neurodegeneration

A common characteristic of neurodegenerative diseases like Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD) is the accumulation of protein aggregates. This reflects a severe disturbance of protein homeostasis, the proteostasis. Here, we review the involvement of the two major proteolytic machineries, the ubiquitin proteasome system (UPS) and the autophagy/lysosomal system, in the pathogenesis of neurodegenerative diseases. These proteolytic systems cooperate to maintain the proteostasis, as is indicated by intricate cross talk. In addition, the UPS and autophagy are regulated by stress pathways that are activated by disturbed proteostasis, like the unfolded protein response (UPR). We will specifically discuss how these proteolytic pathways are affected in neurodegenerative diseases. We will show that there is a differential involvement of the UPS and autophagy in different neurodegenerative disorders. In addition, the proteolytic impairment may be primary or secondary to the pathology. These differences have important implications for the design of therapeutic strategies. The opportunities and caveats of targeting the UPS and autophagy/lysosomal system as a therapeutic strategy in neurodegeneration will be discussed.     

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.     

The role of ubiquitin C-terminal hydrolase L1 in neurodegenerative disorders

Impairment of the ubiquitin-proteasome system (UPS) results in the failure to remove and degrade misfolded proteins and consequently causes the accumulation of misfolded proteins in the cell. The aberrant interactions between misfolded proteins and normal intracellular proteins are thought to underlie the pathogenesis in many neurodegenerative diseases. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an important component of the UPS. Its major function is related to mono-ubiquitin recycling and thereby, sustaining protein degradation. Mutations of the UCH-L1 gene and alterations of its proteins' activity have been found to associate with several neurodegenerative disorders. In this review, we will discuss a link between UCH-L1 and Parkinson's, Huntington's and Alzheimer's diseases. We will also present a potential strategy for the treatment of Alzheimer's disease by boosting endogenous UCH-L1 activity.     

The many roles of chemokine receptors in neurodegenerative disorders: emerging new therapeutical strategies

Chemokines and chemokine receptors, primarily found to play a role in leukocyte migration to the inflammatory sites or to second lymphoid organs, have recently been found expressed on the resident cells of the central nervous system (CNS). These proteins are important for the development of the CNS and are involved in normal brain functions such as synaptic transmission. Increasing lines of evidence have implicated an involvement for chemokines and their receptors in several neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), human immunodeficiency virus-associated dementia (HAD), multiple sclerosis (MS), and stroke. Specific inhibition of the biological activities of chemokine receptors could gain therapeutic benefit for these neurodegenerative disorders. In recent years, non-peptide antagonists of chemokine receptors have been disclosed and tested in relevant pharmacological models and some of these inhibitors have entered clinical trials. The aim of this review is to outline the recent progress regarding the role of chemokines and their receptors in neurodegenerative diseases and the advancements in the development of chemokine receptor inhibitors as potential therapeutic approaches for these neurodegenerative diseases.     

A novel method for evaluation and screening of caspase inhibitory peptides by the amino acid positional fitness score

Background

Since caspases are key executioners of apoptosis in cases of severe diseases including neurodegenerative disorders such as Alzheimer's disease and Huntington's disease, and viral infection diseases such as AIDS and hepatitis, potent and specific inhibitors of caspases have clinical potential. A series of peptide inhibitors has been designed based on cleavage sites of substrate proteins. However, these peptides are not necessarily the most potent to each caspase. Moreover, so far, it has proved to be difficult to design potent and specific peptide inhibitors of each caspase from sequence data of known cleavage sites in substrate proteins. We have attempted to develop a computational screening system for rapid selection of potent and specific peptide inhibitors from a comprehensive peptide library.

Results

We developed a new method for rapid evaluation and screening of peptide inhibitors based on Amino acid Positional Fitness (APF) score. By using this score, all known peptide inhibitors of each caspases-3,-7,-8, and -9 were rapidly selected in their enriched libraries. In this libraries, there were good correlations between predicted binding affinities of the known peptide inhibitors and their experimental Ki values. Furthermore, a novel potent peptide inhibitor, Ac-DNLD-CHO, for caspase-3 was able to be designed by this method. To our knowledge, DNLD is a first reported caspase-3 inhibitory peptide identified by using the computational screening strategy.

Conclusion

Our new method for rapid screening of peptide inhibitors using APF score is an efficient strategy to select potent and specific peptide inhibitors from a comprehensive peptide library. Thus, the APF method has the potential to become a valuable approach for the discovery of the most effective peptide inhibitors. Moreover, it is anticipated that these peptide inhibitors can serve as leads for further drug design and optimization of small molecular inhibitors.     

MAPKs: new targets for neurodegeneration

Neurodegenerative diseases remain a huge unmet pharmaceutical need. For some diseases such as Parkinson's disease, there are currently only palliative therapies, and for others such as Alzheimer's disease there are no proven therapies on the market that have any significant impact on disease progression. Recent work has suggested that cell death may play a key role in a number of neurodegenerative diseases, and halting this aberrant cell death may halt disease progression. Kinases identified in cell death pathways may be attractive targets for neurodegenerative diseases. In this review, the authors will focus on three families of related mitogen-activated protein kinases (MAPKs), namely, the extracellular signal-regulated protein kinases (ERKs), the c-Jun N-terminal kinases (JNKs) and the p38 MAPKs. The evidence for activation of each of these pathways in disease states and in models of neurodegenerative disorders will be examined. Effects of inhibitors, where available, will be discussed, and potential problems and side effects of kinase inhibitors as therapeutics will be addressed.     

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.     

Transgenic animal models of neurodegenerative diseases and their application to treatment development

Neurodegenerative disorders of the aging population affect over 5 million people in the US and Europe alone. The common feature is the progressive accumulation of misfolded proteins with the formation of toxic oligomers. Previous studies show that while in Alzheimer's disease (AD) misfolded amyloid-beta protein accumulates both in the intracellular and extracellular space, in Lewy body disease (LBD), Parkinson's disease (PD), Multiple System Atrophy (MSA), Fronto-Temporal dementia (FTD), prion diseases, amyotrophic lateral sclerosis (ALS) and trinucleotide repeat disorders (TNRD), the aggregated proteins accumulate in the plasma membrane and intracellularly. Protein misfolding and accumulation is the result of an altered balance between protein synthesis, aggregation rate and clearance. Based on these studies, considerable advances have been made in the past years in developing novel experimental models of neurodegenerative disorders. This has been in part driven by the identification of genetic mutations associated with familial forms of these conditions and gene polymorphisms associated with the more common sporadic variants of these diseases. Transgenic and knock out rodents and Drosophila as well as viral vector driven models of Alzheimer's disease (AD), PD, Huntington's disease (HD) and others have been developed, however the focus for this review will be on rodent models of AD, FTD, PD/LBD, and MSA. Promising therapeutic results have been obtained utilizing amyloid precursor protein (APP) transgenic (tg) models of AD to develop therapies including use of inhibitors of the APP-processing enzymes beta- and gamma-secretase as well as vaccine therapies.     

Therapeutic induction of autophagy to modulate neurodegenerative disease progression

There is accumulating evidence that aggregating, misfolded proteins may have an impact on autophagic function, suggesting that this could be a secondary pathological mechanism in many diseases. In this review, we focus on the role of autophagy in four major neurodegenerative diseases: Alzheimer disease (AD), Huntington''s disease (HD), Parkinson''s disease (PD) and amyotropic lateral sclerosis.     

Phytochemicals Targeting Oxidative Stress,Interconnected Neuroinflammatory,and Neuroapoptotic Pathways Following Radiation

The radiation for therapeutic purposes has shown positive effects in different contexts; however, it can increase the risk of many age-related and neurodegenerative diseases such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and Parkinson’s disease (PD). These different outcomes highlight a dose-response phenomenon called hormesis. Prevailing studies indicate that high doses of radiation could play several destructive roles in triggering oxidative stress, neuroapoptosis, and neuroinflammation in neurodegeneration. However, there is a lack of effective treatments in combating radiation-induced neurodegeneration, and the present drugs suffer from some drawbacks, including side effects and drug resistance. Among natural entities, polyphenols are suggested as multi-target agents affecting the dysregulated pathogenic mechanisms in neurodegenerative disease. This review discusses the destructive effects of radiation on the induction of neurodegenerative diseases by dysregulating oxidative stress, apoptosis, and inflammation. We also describe the promising effects of polyphenols and other candidate phytochemicals in preventing and treating radiation-induced neurodegenerative disorders, aiming to find novel/potential therapeutic compounds against such disorders.     

Antiapoptotic drugs: a therapautic strategy for the prevention of neurodegenerative diseases

The purpose of this review is to discuss potential pathways involved in the pathogenesis of neurodegenerative diseases, highlighting current pharmacological drug targets in neuronal apoptosis prevention. The incidence of these disorders is expected to rise in the coming years and so finding effective treatments represents a significant challenge for medicine. Alzheimer's disease and Parkinson's disease were both described almost a century ago and are the most important neurodegenerative disorders in the developed world. However, the molecular mechanisms that lead to the development of the neuronal pathology in both diseases are unclear. For this reason, despite substantial research in the area, an effective treatment for these diseases does not yet exist. In the present study we discuss in depth the pathways involved in apoptosis and neuronal death in neurodegenerative diseases. We also examine drugs that may have a neuroprotective effect. Inhibition of apoptosis mediated by oxidative stress generation and mitochondrial alteration or by the blockade of NMDA receptors could constitute a suitable therapeutic strategy for Alzheimer's disease. A multiple therapy with antioxidants, cell cycle inhibitors, GSK3β inhibitors, and STATINS could, in the future, represent a suitable strategy for delaying the progression of neurodegenerative diseases. This research contributes to the development of new methods in the field of apoptosis inhibitors that could provide the future tools for the treatment of Alzheimer's and Parkinson's disease, as well as other neurodegenerative diseases.