Molecular clocks

A convenient and precise mass spectrometric method for measurement of the deamidation rates of glutaminyl and asparaginyl residues in peptides and proteins has been developed; the rates of deamidation of 306 asparaginyl sequences in model peptides at pH 7.4, 37.0 degrees C, 0.15 M Tris.HCl buffer have been determined; a library of 913 amide-containing peptides for use by other investigators in similar studies has been established; and, by means of simultaneous deamidation rate measurements of rabbit muscle aldolase and appropriate model peptides in the same solutions, the use of this method for quantitative measurement of the relative effects of primary, secondary, tertiary, and quaternary protein structure on deamidation rates has been demonstrated. The measured rates are discussed with respect to the hypothesis that glutaminyl and asparaginyl residues serve, through deamidation, as molecular timers of biological events.     

Age-dependent deamidation of asparagine residues in proteins

Nonenzymatic deamidation of peptides and proteins represents an important degradation reaction occurring in vitro in the course of isolation or storage and in vivo during development and/or aging of cells. This review first presents a synopsis of the influence of structure on deamidation reaction proceeding via a five-membered succinimide intermediate, followed by an outline of procedures for separation and detection of deamidated forms. Selected examples for in vitro and in vivo deamidation are reviewed including the possible biological consequences of this protein degradation. Finally, the reaction of protein methyltransferase with L-isoaspartyl- and D-aspartyl residues and its possible role in protein repair is elucidated.     

Distribution of glutamine and asparagine residues and their near neighbors in peptides and proteins.

In a statistical study of neighboring residues in 1465 peptides and proteins comprising 450,431 residues, it was found that the preferences for residues neighboring to glutamine and asparagine residues are consistent with the hypothesis that the rates of deamidation of these residues are of biological significance. Some dipeptide and tripeptide structures have special usefulness and some are especially undesirable. More such structures exist for amide residues than for other residues, and their specific types are those most relevant to the deamidation of amide residues under biological conditions.     

Deamidation of human erythrocyte protein 4.1: possible role in aging.

The human erythrocyte membrane protein 4.1 exists in two major electrophoretic forms: 4.1a (80 Kd) and 4.1b (78 Kd). Mass spectrometry and amino acid analysis of the proteolytic peptides derived from carboxyl-terminal regions of these proteins indicate that they differ by deamidation of two aspargine residues at positions 478 and 502. Electrophoretic analysis of carboxyl-terminal peptides has shown that the mobility difference between the two polypeptides is due to the deamidation of Asn502 and not that of Asn478. This observation was confirmed by converting a congener of the protein 4.1b to 4.1a by site-directed mutagenesis of Asn502 to Asp. These results unambiguously demonstrate that deamidation of Asn502 is responsible for conversion of protein 4.1b to 4.1a. Since the conversion of protein 4.1b to 4.1a, under physiological conditions, occurs in a time-dependent manner, our study clearly shows that deamidation is an excellent marker for red blood cell aging.     

Controlled Deamidation of Peptides and Proteins: An Experimental Hazard and a Possible Biological Timer

Experiments on model peptides show that the rate of deamidation of asparaginyl residues depends strongly on the nature of neighboring residues. The natural distribution of glutaminyl and asparaginyl residues is ordered with respect to the biological lifetime of the peptides and the functional groups of the residues neighboring to glutaminyl and asparaginyl residues. The rates of deamidation of such amide peptides under physiological conditions could serve as useful timers of development and aging.     

Protein deamidation

A completely automatic computerized technique for the quantitative estimation of the deamidation rates of any protein for which the three-dimensional structure is known has been developed. Calculations of the specific deamidation rates of 170,014 asparaginyl residues in 13,335 proteins have been carried out. The calculated values have good quantitative reliability when compared with experimental measurements. These rates demonstrate that deamidation may be a biologically relevant phenomenon in a remarkably large percentage of proteins.     

Prediction of protein deamidation rates from primary and three-dimensional structure

A method for the quantitative estimation of instability with respect to deamidation of the asparaginyl (Asn) residues in proteins is described. The procedure involves the observation of several simple aspects of the three-dimensional environment of each Asn residue in the protein and a calculation that includes these observations, the primary amino acid residue sequence, and the previously reported complete set of sequence-dependent rates of deamidation for Asn pentapeptides. This method is demonstrated and evaluated for 23 proteins in which 31 unstable and 167 stable Asn residues have been reported and for 7 unstable and 63 stable Asn residues that have been reported in 61 human hemoglobin variants. The relative importance of primary structure and three-dimensional structure in Asn deamidation is estimated.     

Deamidation of human proteins

Deamidation of asparaginyl and glutaminyl residues causes time-dependent changes in charge and conformation of peptides and proteins. Quantitative and experimentally verified predictive calculations of the deamidation rates of 1,371 asparaginyl residues in a representative collection of 126 human proteins have been performed. These rates suggest that deamidation is a biologically relevant phenomenon in a remarkably large percentage of human proteins.     

Deamidation In Vivo of an Asparagine Residue of Rabbit Muscle Aldolase

Microheterogeneity of rabbit muscle aldolase is caused by deamidation in vivo of an asparagine residue near the C-terminus of each subunit. Isotopic labeling of a peptide containing the asparagine residue at various time intervals before isolation of aldolase permits estimation of the half-time for the deamidation as about 8 days, which is about the time estimated for the half-life of the enzyme in vivo. It is concluded that the aldolase as genetically determined is a tetramer, designated alpha(4), that undergoes random deamidation to form alpha(3)beta, alpha(2)beta(2), and alphabeta(3) species as intermediates in the formation of beta(4), the species in which all of the specific asparagine has been deamidated. Isoelectric focusing data indicate that the subunits do not exchange appreciably in vivo.     

Evolution and the Distribution of Glutaminyl and Asparaginyl Residues in Proteins

Recent experiments on the deamidation of glutaminyl and asparaginyl residues in peptides and proteins support the hypothesis that these residues may serve as molecular clocks that control biological processes. A hypothesis is now offered that suggests that these molecular clocks are set by rejection or accumulation of appropriate sequences of residues including a glutaminyl or asparaginyl residue during evolution.     

Asparaginyl deamidation-methylation of rat ventricular myosin light chains

Spontaneous asparaginyl deamidation can produce damage to cytoskeletal proteins, and may lead to their targeting for subsequent rapid intracellular breakdown or repair. To test if myofibrillar proteins are subject to spontaneous deamidation damage in vitro, purified rat ventricular myosin light chain 1 (MLC1v) and phosphorylatable myosin light chain 2 (MPLC2v) were incubated (37 degrees C, 4 h, pH 2-11), and tested as substrates for human erythrocyte and rat cardiac protein carboxyl methyltransferase (PCMT). PCMT catalyzes the transfer of a methyl group from [3H-methyl] S-adenosyl methionine to deamidated asparaginyl residues and altered aspartyl residues on damaged proteins. MLC1v and MPLC2v underwent extensive incubation damage at neutral and alkaline pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography revealed 3H-incorporation into MLC1v, MPLC2v, and a Mr = 14,000 polypeptide. 3H-methylated, CNBr-cleavage fragments of PCMT-methylated light chains were then separated by reverse-phase high performance liquid chromatography, and sequenced by automated Edman degradation. The major 3H-labeled peptide of the Mr = 14,000 protein proved homologous to residues 84 to 104 of rat MPLC2v, with a proposed deamidation site at Asn99-Ala100. The major 3H-labeled peptide from MLC1v proved homologous to residues 73 to 111 of rat cardiac MLC1v, with a proposed deamidation site at Asn108-Ser109. These results indicate that both myofibrillar protein subunits undergo selective non-enzymatic degradation at neutral and alkaline pH, resulting in the formation of methyl acceptor sites for human erythrocyte and rat cardiac PCMT. PCMT-catalyzed methylation of ventricular myosin light chains may be important in the repair, or subsequent proteolysis of these long-lived structural proteins of the myofibril.     

Immunoreactivity of Antibodies Against Transglutaminase-Deamidated Gliadins in Adult Celiac Disease

Background The significance of the presence of anti-gliadin antibodies in patients affected by celiac disease is still unclear. It is hypothesized that gliadin deamidation, catalysed by transglutaminase, plays a role in favoring the antigen presentation. Aim To determine the immunoreactivity of anti-gliadin antibodies from untreated celiac patients to transglutaminase deamidated gliadins. Materials and methods Gliadins from wheat flour underwent enzymatic digestion and were deamidated or cysteamine-transamidated by transglutaminase. Immunoreactivity of anti-gliadin antibodies from untreated adult celiac patients sera was evaluated by means of a competitive enzyme-linked immunosorbent assay (ELISA) method. Results Gliadin deamidation increased antibodies immunoreactivity from 25% to 50% while cysteamine incorporation into the gliadin peptides resulted in an immunoreactivity decrease. Conclusions Increased immunoreactivity of transglutaminase deamidated gliadins tested with anti-gliadin antibodies from untreated adult celiac patients supports the hypothesis of a pivotal role of gliadin deamidation in the pathomechanism of celiac disease.     

Structural mechanism of ubiquitin and NEDD8 deamidation catalyzed by bacterial effectors that induce macrophage-specific apoptosis

Targeting eukaryotic proteins for deamidation modification is increasingly appreciated as a general bacterial virulence mechanism. Here, we present an atomic view of how a bacterial deamidase effector, cycle-inhibiting factor homolog in Burkholderia pseudomallei (CHBP), recognizes its host targets, ubiquitin (Ub) and Ub-like neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), and catalyzes site-specific deamidation. Crystal structures of CHBP–Ub/NEDD8 complexes show that Ub and NEDD8 are similarly cradled by a large cleft in CHBP with four contacting surfaces. The pattern of Ub/NEDD8 recognition by CHBP resembles that by the E1 activation enzyme, which critically involves the Lys-11 surface in Ub/NEDD8. Close examination of the papain-like catalytic center reveals structural determinants of CHBP being an obligate glutamine deamidase. Molecular-dynamics simulation identifies Gln-31/Glu-31 of Ub/NEDD8 as one key determinant of CHBP substrate preference for NEDD8. Inspired by the idea of using the unique bacterial activity as a tool, we further discover that CHBP-catalyzed NEDD8 deamidation triggers macrophage-specific apoptosis, which predicts a previously unknown macrophage-specific proapoptotic signal that is negatively regulated by neddylation-mediated protein ubiquitination/degradation.     

Secreted mouse prolactin (PRL) and stored ovine PRL. II. Role of amides in receptor binding and immunoreactivity

Native PRL and des-amido forms 1, 2, and 3 were tested for their individual immunochemical and receptor-binding abilities. The results show that deamidation of either secreted mouse PRL or stored ovine PRL alters their binding in a radioreceptor assay. For each accumulated deamidation there was a statistically significant (P less than 0.05) decrease in the binding potency of either stored ovine PRL or secreted mouse PRL to a cell membrane receptor preparation. RIAs indicated that there was a statistically significant (P less than 0.05) decrease in PRL's immunopotency toward polyclonal antisera only when select residues deamidated. This study suggests that all the asn/gln residues which deamidate in order to make des-amido forms 1, 2, and 3 constitute part of the receptor-binding domains of both secreted and stored PRLs, while only a fraction of those same residues form portions of PRL's antigenic sites. Thus PRL's receptor-binding surface is separated from its antigenic sites, with only partial overlap being indicated. Our data also indicate that there are no major structural differences in the receptor-binding domains of secreted and stored PRLs. We also see that the receptor-binding domain of PRL has been highly conserved throughout evolution. Since the binding affinity of PRL to a membrane-bound receptor can be altered through deamidation, we view the process as a plausible regulatory mechanism for controlling the quantitative action of PRL at a given target organ.     

Enzymatic deamidation of methyl-accepting chemotaxis proteins in Escherichia coli catalyzed by the cheB gene product.

The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli undergo reversible methylation that has been correlated with adaptation of cells to environmental stimuli. MCPI, the product of the tsr gene, accepts methyl groups at multiple sites that are located on two tryptic peptides, denoted K1 and R1. A second modification of the MCPs, which is not methylation, has been designated the CheB-dependent modification. A CheB-dependent modification occurs on methyl-accepting peptide K1 and allows additional methyl groups to be incorporated into this peptide. We have performed partial amino acid sequence analyses on radiolabeled peptides K1 and R1 derived from MCPI and have identified several methyl-accepting sites. We found that, in the absence of CheB-dependent modification, a site in peptide K1 is unable to accept methyl groups. Correlation of this protein sequence data with the nucleotide sequence of the tsr gene [Boyd, A., Kendall, K. & Simon, M.I. (1983) Nature (London) 301, 623-626] suggests that CheB-dependent modification of MCPI is the enzymatic deamidation of glutamine to methyl-accepting glutamic acid. Possible roles for this deamidation in bacterial chemotaxis are discussed.     

The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides

BACKGROUND & AIMS: Gluten (GLU)-specific T-cell responses in HLA-DQ2 positive adult celiac disease (CD) patients are directed to an immunodominant alpha-gliadin (GLIA) peptide that requires deamidation for T-cell recognition. The aim of the current study was to determine which GLU peptide(s) are involved early in disease. METHODS: We have characterized the GLU-specific T-cell response in HLA-DQ2 positive children with recent onset CD. RESULTS: We found that 50% of these patients do not respond to the alpha-GLIA peptide but to a diverse set of GLIA and glutenin (GLT) peptides, including 6 novel epitopes. Moreover, individual patients respond to distinct (combinations of) GLU peptides. T-cell cross-reactivity toward homologous GLIA and GLT peptides was observed, which might play a role in the initial spreading of the GLU-specific T-cell response. Although all pediatric patients displayed deamidation-dependent responses, deamidation-independent responses were found in the majority of patients as well. Finally, T-cell responses to 3 of these novel GLU peptides were found in adult CD patients. CONCLUSIONS: The diversity of the GLU-specific T-cell response is far greater than was previously appreciated. Both adult and young CD patients can respond to a diverse repertoire of GLU peptides. The observation that T-cell responses to 3 of the novel peptides are independent of deamidation indicates that T-cell responses can be initiated toward native GLU peptides. The possibility that deamidation drives the GLU response toward immunodominant T-cell stimulatory peptides after disease initiation is discussed.     

Bordetella bronchiseptica dermonecrotizing toxin induces reorganization of actin stress fibers through deamidation of Gln-63 of the GTP-binding protein Rho

Bordetella dermonecrotizing toxin causes assembly of actin stress fibers and focal adhesions in some cultured cells and induces mobility shifts of the small GTP-binding protein Rho on electrophoresis. We attempted to clarify the molecular basis of the toxin action on Rho. Analysis of the amino acid sequence of toxin-treated RhoA revealed the deamidation of Gln-63 to Glu. The substitution of Glu for Gln-63 of RhoA by site-directed mutagenesis caused a mobility shift on electrophoresis, which was indistinguishable from that of the toxin-treated RhoA. Neither mutant RhoA-bearing Glu-63 nor toxin-treated RhoA significantly differed from untreated wild type RhoA in guanosine 5′-[γ-thio]triphosphate binding activity but both showed a 10-fold reduction in GTP hydrolysis activity relative to untreated RhoA. C3H10T1/2 cells transfected with cDNA of the mutant RhoA bearing Glu-63 showed extensive formation of actin stress fibers similar to the toxin-treated cells. These results indicate that the toxin catalyzes deamidation of Gln-63 of Rho and renders it constitutively active, leading to formation of actin stress fibers.     

Role of p38alpha MAPK in cardiac apoptosis and remodeling after myocardial infarction

Acute coronary occlusion results in ischemia-mediated death of cardiomyocytes. In the days and weeks following myocardial infarction (MI), left ventricular remodeling occurs that is characterized by persistent cardiomyocyte apoptosis, thinning and fibrosis at the site of infarction, ventricular chamber dilatation, and growth of remaining viable cardiomyocytes. The p38 mitogen-activated protein kinase (MAPK) signaling cascade has been implicated in the remodeling process. In this work, mice with cardiac-specific expression of a dominant negative mutant form of p38 MAPK (DN-p38alpha) were subjected to MI by occlusion of the left coronary artery. Acute ischemia area was determined by transthoracic echocardiography 2 h after MI surgery, and was found to be nearly identical in DN-p38 mice and their wild-type littermates. Seven days after MI, mice were subjected to repeat echocardiography and histological examination of infarct size. DN-p38 mice had markedly reduced infarct size and increased ventricular systolic function 7 days after MI when compared to wild-type littermates. In addition, DN-p38 mice had less cardiomyocyte apoptosis than wild-type mice in the infarct border zone. Recently, it was discovered that Bcl-X(L) deamidation occurs in vivo, and this results in Bcl-X(L) degradation that sensitizes cells to apoptosis by enhancing BAX activity. Bcl-X(L) deamidation was found to occur in the cardiac tissue of wild-type mice after MI, but was reduced in DN-p38 mice. These results establish that p38 MAPK activity is required for pathological remodeling after MI and suggest that p38 MAPK may promote cardiomyocyte apoptosis through Bcl-X(L) deamidation.     

Alkaline phosphatase dissolves calcium pyrophosphate dihydrate crystals.

We have shown that yeast pyrophosphatase dissolves calcium pyrophosphate dihydrate (CPPD) crystals in solutions. In this investigation we demonstrate that alkaline phosphatase (ALP) effectively dissolves CPPD crystals in vitro. CPPD dissolution by ALP had a pH optimum of 7.4, which is the optimum pH for its pyrophosphatase (PPiase) activity. The CPPD dissolution and PPiase activity by ALP are magnesium dependent, whereas its phosphoester hydrolytic activity is not. Calcium, which inhibited the enzymatic CPPD dissolution and PPiase activity of ALP had no effect on its phosphoester hydrolytic activity. These data indicate that PPiase activity of ALP is responsible for CPPD dissolution and not its phosphoester hydrolytic activity. Matrix molecules such as proteoglycans and chondroitin sulfate had no effect on the enzymatic and nonenzymatic dissolution of CPPD crystals. ALP acted more effectively on CPPD crystals than on soluble pyrophosphate relative to yeast PPiase. Our data suggest that chondrocyte ALP may play an important role in the dissolution of CPPD crystals in cartilage.     

Transglutaminases in inflammation and fibrosis of the gastrointestinal tract and the liver

Transglutaminases are a family of eight currently known calcium-dependent enzymes that catalyze the cross-linking or deamidation of proteins. They are involved in important biological processes such as wound healing, tissue repair, fibrogenesis, apoptosis, inflammation and cell-cycle control. Therefore, they play important roles in the pathomechanisms of autoimmune, inflammatory and degenerative diseases, many of which affect the gastrointestinal system.Transglutaminase 2 is prominent, since it is central to the pathogenesis of celiac disease, and modulates inflammation and fibrosis in inflammatory bowel and chronic liver diseases.This review highlights our present understanding of transglutaminase function in gastrointestinal and liver diseases and therapeutic strategies that target transglutaminase activities.