Physio-pathological roles of transglutaminase-catalyzed reactions

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze post-translational modifications of proteins. The main activity of these enzymes is the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted /crosslinked adducts) or -OH groups (to form ester linkages). In the absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. The TG enzymes are also capable of catalyzing other reactions important for cell viability. The distribution and the physiological roles of TG enzymes have been widely studied in numerous cell types and tissues and their roles in several diseases have begun to be identified. "Tissue" TG (TG2), a member of the TG family of enzymes, has definitely been shown to be involved in the molecular mechanisms responsible for a very widespread human pathology: i.e. celiac disease (CD). TG activity has also been hypothesized to be directly involved in the pathogenetic mechanisms responsible for several other human diseases, including neurodegenerative diseases, which are often associated with CD. Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, supranuclear palsy, Huntington's disease and other recently identified polyglutamine diseases, are characterized, in part, by aberrant cerebral TG activity and by increased cross-linked proteins in affected brains. In this review, we discuss the physio-pathological role of TG-catalyzed reactions, with particular interest in the molecular mechanisms that could involve these enzymes in the physio-pathological processes responsible for human neurodegenerative diseases.     

Increased levels of gamma-glutamylamines in Huntington disease CSF

Transglutaminases (TGases) catalyze several reactions with protein substrates, including formation of γ-glutamyl-ε-lysine cross-links and γ-glutamylpolyamine residues. The resulting γ-glutamylamines are excised intact during proteolysis. TGase activity is altered in several diseases, highlighting the importance of in situ enzymatic determinations. Previous work showed that TGase activity (as measured by an in vitro assay) and free γ-glutamyl-ε-lysine levels are elevated in Huntington disease (HD) and that γ-glutamyl-ε-lysine is increased in HD CSF. Although free γ-glutamyl-ε-lysine was used in these studies as an index of in situ TGase activity, γ-glutamylpolyamines may also be diagnostic. We have devised methods for the simultaneous determination of four γ-glutamylamines in CSF: γ-glutamyl-ε-lysine, γ-glutamylspermidine, γ-glutamylputrescine, and bis-γ-glutamylputrescine and showed that all are present in normal human CSF at concentrations of ∼150, 670, 40, and 240 nM, respectively. The high γ-glutamylspermidine/γ-glutamylputrescine and γ-glutamylspermidine/bis-γ-glutamylputrescine ratios presumably reflect in part the large spermidine to putrescine mole ratio in human brain. We also showed that all four γ-glutamylamines are elevated in HD CSF. Our findings support the hypotheses that (i) γ-glutamylpolyamines are reflective of TGase activity in human brain, (ii) polyamination is an important post-translational modification of brain proteins, and (iii) TGase-catalyzed modification of proteins is increased in HD brain.     

Transglutaminase inhibition: A therapy to protect cells from death in neurodegeneration?

Transglutaminases(TGs;E.C.2.3.2.13)are ubiquitous enzymes which catalyze post-translational modifications of proteins.TGs and TG-catalyzed post-translational modifications of proteins have been shown to be involved in the molecular mechanisms responsible for several human diseases.In particular,TG activity has been hypothesized to also be involved also in the molecular mechanisms responsible for human neurodegenerative diseases.In support of this hypothesis,Basso et al recently demonstrated that the TG inhibition protects against oxidative stress-induced neuronal death,suggesting that multiple TG isoforms participate in oxidative stress-induced cell death and that nonselective TG isoform inhibitors will be most effective in fighting oxidative death in neurological disorders.In this commentary,we discuss the possible molecular mechanisms by which TG activity could be involved in the pathogenesis of neurological diseases,with particular reference to neurodegenerative diseases,and the possible involvement of multiple TG isoforms expressed simultaneously in the nervous system in these diseases.Moreover,therapeutic strategies based on the use of selective or nonselective TG inhibitors for the amelioration of thesymptoms of patients with neurological diseases,characterized by aberrant TG activity,are also discussed.     

Recent advances in the development of tissue transglutaminase (TG2) inhibitors

Tissue transglutaminase (TG2) is a Ca²⁺-dependent enzyme and probably the most ubiquitously expressed member of the mammalian transglutaminase family. TG2 plays a number of important roles in a variety of biological processes. Via its transamidating function, it is responsible for the cross-linking of proteins by forming isopeptide bonds between glutamine and lysine residues. Intracellularly, Ca²⁺ activation of the enzyme is normally tightly regulated by the binding of GTP. However, upregulated levels of TG2 are associated with many disease states like celiac sprue, certain types of cancer, fibrosis, cystic fibrosis, multiple sclerosis, Alzheimer’s, Huntington’s and Parkinson’s disease. Selective inhibitors for TG2 both cell penetrating and non-cell penetrating would therefore serve as novel therapeutic tools for the treatment of these disease states. Moreover, they would provide useful tools to fully elucidate the cellular mechanisms TG2 is involved in and help comprehend how the enzyme is regulated at the cellular level. The current paper is intended to give an update on the recently discovered classes of TG2 inhibitors along with their structure–activity relationships. The biological properties of these derivatives, in terms of both activity and selectivity, will also be reported in order to translate their potential for future therapeutic developments.     

Epigenetic Modulation in Parkinson’s Disease and Potential Treatment Therapies

In the recent past, huge emphasis has been given to the epigenetic alterations of the genes responsible for the cause of neurological disorders. Earlier, the scientists believed somatic changes and modifications in the genetic makeup of DNA to be the main cause of the neurodegenerative diseases. With the increase in understanding of the neural network and associated diseases, it was observed that alterations in the gene expression were not always originated by the change in the genetic sequence. For this reason, extensive research has been conducted to understand the role of epigenetics in the pathophysiology of several neurological disorders including Alzheimer’s disease, Parkinson’s disease and, Huntington’s disease. In a healthy person, the epigenetic modifications play a crucial role in maintaining the homeostasis of a cell by either up-regulating or down-regulating the genes. Therefore, improved understanding of these modifications may provide better insight about the diseases and may serve as potential therapeutic targets for their treatment. The present review describes various epigenetic modifications involved in the pathology of Parkinson’s Disease (PD) backed by multiple researches carried out to study the gene expression regulation related to the epigenetic alterations. Additionally, we will briefly go through the current scenario about the various treatment therapies including small molecules and multiple phytochemicals potent enough to reverse these alterations and the future directions for a better management of PD.

     

Characterization of sea urchin transglutaminase, a protein regulated by guanine/adenine nucleotides

Transglutaminases (TGs) are calcium-dependent enzymes that catalyze the transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGs have been identified in mammals, and three of them (types 2, 3, and 5) are regulated by GTP/ATP and are able to hydrolyze GTP, working as bifunctional enzymes. We have isolated a cDNA clone encoding a TG from a cDNA library prepared from the blastula stage of sea urchin Paracentrotus lividus (PlTG). The cDNA sequence has an open reading frame coding for a protein of 738 amino acids, including a Cys active site and two other residues critical for catalytic activity, His and Asp. We have studied its expression pattern by in situ hybridization and have also demonstrated that the in vitro expressed PlTG had GTP- and ATP-hydrolyzing activity; moreover, GTP inhibited the transamidating activity of this enzyme as it does that of human TG2, TG3, and TG5.     

Evidences for a role of protein cross-links in transglutaminase-related disease

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. Considerable and intense progress has been made in the understanding of the chemistry, molecular biology and cell biology of TGs. The knowledge that very different physiological and pathological processes are dependent on the presence of adequate levels of these cross-linking enzymes and on the amount of both free and protein-conjugated polyamines by TG, has generated an incredible amount of original research and review articles. It is clear that TG-mediated reactions are essential for some biological processes, such as blood coagulation, skin barrier formation and extracellular matrix assembly, but may also be involved in pathogenetic mechanisms responsible for several human diseases, such as cancer, AIDS, neurodegenerative disorders, celiac disease, and eye lens opacification. We present here a comprehensive review of recent insights into the pathophysiology of TGs related to their protein cross-linking activity.     

Polyglutamine misfolding in yeast: Toxic and protective aggregation

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.Key words: polyglutamine proteins, neurodegeneration, aggresome, Huntington disease, yeast models     

多巴胺与神经退行性疾病研究进展

多巴胺调控人类的情绪和认识能力,包括思想、感觉、理解、推理等,同时,它也在人类的运动功能中发挥重要作用。研究表明多巴胺的合成、储存、释放、降解和重摄取等失衡均与中枢神经系统的多种退行性疾病有密切联系,同时许多治疗疾病的有效药物也围绕多巴胺的研究而产生,如多巴胺替代疗法改善帕金森病的运动症状,多巴胺受体阻断剂可改善舞蹈病的运动症状以及调节多种疾病的精神症状,在临床上都取得了可喜的疗效。然而目前未发现与多巴胺代谢直接相关的基因突变,因此未来需要继续深入研究在神经退行性疾病中造成多巴胺代谢失常的机制,旨在为临床新药物靶点和新治疗手段的研发提供线索。     

Quantitative Proteomic Analysis of Human Substantia Nigra in Alzheimer’s Disease, Huntington’s Disease and Multiple Sclerosis

The substantia nigra plays important roles in the brain function and is critical in the development of many diseases, particularly Parkinson??s disease. Pathological changes of the substantia nigra have also been reported in other neurodegenerative diseases. Using a quantitative proteomic approach, we investigated protein expressions in the substantia nigra of Alzheimer??s disease, Huntington??s disease, and Multiple sclerosis. The expression level of one hundred and four proteins that were identified in at least three samples of each group were compared with the control group, with nineteen, twenty-two and thirteen proteins differentially expressed in Alzheimer??s diseases, Huntington??s disease and Multiple sclerosis respectively. The result indicates that the substantia nigra also undergoes functional adaption or damage in these diseases.     

A nonradioactive dot blot assay for transglutaminase activity

Aberrant transglutaminase (TG) activity has been implicated in the pathology of numerous diseases, including Huntington’s disease and Alzheimer’s disease. To fully characterize the role of TGs in these disorders, it is important that simple quantifiable assays be made available. The most commonly used assay currently employed requires significant time and a radioactive substrate. The assay described here uses a biotinylated substrate in conjunction with a dot blot apparatus to eliminate the use of radioactive substrates and allows relative transglutaminase activity to be measured simultaneously with minimal sample preparation in a large number of samples containing purified enzyme, cell extracts, or tissue homogenates.     

Apolipoprotein E content of VLDL limits LPL-mediated triglyceride hydrolysis

High levels of circulating triglycerides (TGs), or hypertriglyceridemia, are key components of metabolic diseases, such as type 2 diabetes, metabolic syndrome, and CVD. As TGs are carried by lipoproteins in plasma, hypertriglyceridemia can result from overproduction or lack of clearance of TG-rich lipoproteins (TRLs) such as VLDLs. The primary driver of TRL clearance is TG hydrolysis mediated by LPL. LPL is regulated by numerous TRL protein components, including the cofactor apolipoprotein C-II, but it is not clear how their effects combine to impact TRL hydrolysis across individuals. Using a novel assay designed to mimic human plasma conditions in vitro, we tested the ability of VLDL from 15 normolipidemic donors to act as substrates for human LPL. We found a striking 10-fold difference in hydrolysis rates across individuals when the particles were compared on a protein or a TG basis. While VLDL TG contents moderately correlated with hydrolysis rate, we noticed substantial variations in non-apoB proteins within these particles by MS. The ability of LPL to hydrolyze VLDL TGs did not correlate with apolipoprotein C-II content, but it was strongly inversely correlated with apolipoprotein E (APOE) and, to a lesser extent, apolipoprotein A-II. Addition of exogenous APOE inhibited LPL lipolysis in a dose-dependent manner. The APOE3 and (particularly) APOE4 isoforms were effective at limiting LPL hydrolysis, whereas APOE2 was not. We conclude that APOE on VLDL modulates LPL activity and could be a relevant factor in the pathogenesis of metabolic disease.     

Polyglutamine misfolding in yeast: Toxic and protective aggregation

Protein misfolding is associated with many human diseases, including neurodegenerative diseases, such as Alzheimer disease, Parkinson disease and Huntington disease. Protein misfolding often results in the formation of intracellular or extracellular inclusions or aggregates. Even though deciphering the role of these aggregates has been the object of intense research activity, their role in protein misfolding diseases is unclear. Here, I discuss the implications of studies on polyglutamine aggregation and toxicity in yeast and other model organisms. These studies provide an excellent experimental and conceptual paradigm that contributes to understanding the differences between toxic and protective trajectories of protein misfolding. Future studies like the ones discussed here have the potential to transform basic concepts of protein misfolding in human diseases and may thus help to identify new therapeutic strategies for their treatment.     

Proteomic analysis of mitochondrial dysfunction in neurodegenerative diseases

Alzheimer’s, Parkinson’s and Huntington’s disease, and amyotrophic lateral sclerosis are the most relevant neurodegenerative syndromes worldwide. The identification of the etiology and additional factors contributing to the onset and progression of these diseases is of great importance in order to develop both preventive and therapeutic intervention. A common feature of these pathologies is the formation of aggregates, containing mutated and/or misfolded proteins, in specific subsets of neurons, which progressively undergo functional impairment and die. The relationship between protein aggregation and the molecular events leading to neurodegeneration has not yet been clarified. In the last decade, several lines of evidence pointed to a major role for mitochondrial dysfunction in the onset of these pathologies. Here, we review how proteomics has been applied to neurodegenerative diseases in order to characterize the relationship existing between protein aggregation and mitochondrial alterations. Moreover, we highlight recent advances in the use of proteomics to identify protein modifications caused by oxidative stress. Future developments in this field are expected to significantly contribute to the full comprehension of the molecular mechanisms at the heart of neurodegeneration.     

Does Huntingtin play a role in selective macroautophagy?

The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.¹ In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases.     

Variations in Both TG1 and TG2 Isozyme-specific In Situ Activities and Protein Expressions during Mouse Embryonic Development

Transglutaminase (TG) is a family of enzymes that catalyzes cross-linking reactions among proteins. Using fluorescent-labeled highly reactive substrate peptides, we recently developed a system to visualize isozyme-specific in situ enzymatic activity. In the present study, we investigated the in situ activities of TG1 (skin-type) and TG2 (tissue-type) using whole mouse sections of various embryonic developmental stages and neonates. In each case, we also successfully used immunostaining of identical whole mouse sections for protein expression after detection of enzymatic activities. In general, the enzymatic activity was correlated with TG protein expression. However, in some tissues, TG protein expression patterns, which were inconsistent with the enzymatic activities, suggested that inactive TGs were produced possibly by self cross-linking or other modifications. Our method allowed us to simultaneously observe developmental variations in both TG isozyme-specific activities and protein levels in mouse embryonic and neonate tissues.     

Novel therapeutic strategies for the treatment of protein-misfolding diseases

Most proteins in the cell adopt a compact, globular fold that determines their stability and function. Partial protein unfolding under conditions of cellular stress results in the exposure of hydrophobic regions normally buried in the interior of the native structure. Interactions involving the exposed hydrophobic surfaces of misfolded protein conformers lead to the formation of toxic aggregates, including oligomers, protofibrils and amyloid fibrils. A significant number of human disorders (e.g. Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis and type II diabetes) are characterised by protein misfolding and aggregation. Over the past five years, outstanding progress has been made in the development of therapeutic strategies targeting these diseases. Three promising approaches include: (1) inhibiting protein aggregation with peptides or small molecules identified via structure-based drug design or high-throughput screening; (2) interfering with post-translational modifications that stimulate protein misfolding and aggregation; and (3) upregulating molecular chaperones or aggregate-clearance mechanisms. Ultimately, drug combinations that capitalise on more than one therapeutic strategy will constitute the most effective treatment for patients with these devastating illnesses.     

Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices

Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca²⁺-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.