Formation Of Protein Crosslinks Research Articles

Derivation of an Inhalation Reference Concentration Based upon Olfactory Neuronal Loss in Male Rats following Subchronic Acetaldehyde Inhalation

Acetaldehyde inhalation induces neoplastic and nonneoplastic responses in the rodent nasal cavity. This experiment further characterizes the dose-response relationship for nasal pathology, nasal epithelial cell proliferation, and DNA–protein cross-link formation in F-344 rats exposed subchronically to acetaldehyde. Animals underwent whole-body exposure to 0, 50, 150, 500, or 1500 ppm acetaldehyde for 6 h/day, 5 days/wk for up to 65 exposure days. Respiratory tract histopathology was evaluated after 4, 9, 14, 30, and 65 exposure days. Acetaldehyde exposure was not associated with reduced body weight gain or other evidence of systemic toxicity. Histologic evaluation of the nasal cavity showed an increased incidence of olfactory neuronal loss (ONL) following acute to subchronic exposure to ≥ 150 ppm acetaldehyde and increased olfactory epithelial cell proliferation following exposure to 1500 ppm acetaldehyde. The severity of the ONL demonstrated dose-and temporal-dependent behaviors, with minimal effects noted at 150–500 ppm acetaldehyde and moderately severe lesions seen in the highest exposure group, with increased lesion severity and extent as the exposure duration increased. Acetaldehyde exposure was also associated with inflammation, hyperplasia, and squamous metaplasia of the respiratory epithelium. These responses were seen in animals exposed to ≥500 ppm acetaldehyde. Acetaldehyde exposure was not associated with increased DNA–protein cross-link formation in the respiratory or olfactory epithelium. A model of acetaldehyde pharmacokinetics in the nose was used to derive an inhalation reference concentration (RfC) of 0.4 ppm, based on the no-observed-adverse-effect level (NOAEL) of 50 ppm for the nasal pathology seen in this study.

Reactions of Glyceraldehyde 3-Phosphate Dehydrogenase Sulfhydryl Groups with Bis-Electrophiles Produce DNA–Protein Cross-Links but Not Mutations

The environmental contaminant 1,2-dibromoethane and diepoxybutane, an oxidation product of the important industrial chemical butadiene, are bis-functional electrophiles and are known to be mutagenic and carcinogenic. One mechanism by which bis-electrophiles can exert their toxic effects is through the induction of genotoxic and mutagenic DNA-peptide cross-links. This mechanism has been shown in systems overexpressing the DNA repair protein O6 -alkylguanine DNA-alkyltransferase (AGT) or glutathione S-transferase and involves reactions with nucleophilic cysteine residues. The hypothesis that DNA-protein cross-link formation is a more general mechanism for genotoxicity by bis-electrophiles was investigated by screening nuclear proteins for reactivity with model monofunctional electrophiles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a candidate because of the nucleophilicity of two cysteine residues (Cys152 and Cys246) in reaction screens with model electrophiles (Dennehy, M. K. et al. (2006) Chem. Res. Toxicol. 19, 20-29). Incubation of GAPDH with bis-electrophiles resulted in inhibition of its catalytic activity, but only at high concentrations of diepoxybutane. In vitro assays indicated DNA-GAPDH cross-link formation in the presence of diepoxybutane, and bis-electrophile reactivity at Cys246 was confirmed using mass spectral analysis. In contrast to AGT, overexpression of human GAPDH in Escherichia coli did not enhance mutagenesis by diepoxybutane. We propose that the lack of mutational enhancement is in part due to the inherently lower reactivity of GAPDH toward bis-electrophiles as well as the reduced DNA binding ability relative to AGT, preventing the in vivo formation of DNA-protein cross-links and enhanced mutagenesis.

Histidine and not tyrosine is required for the Peroxide‐induced formation of haem to protein cross‐linked myoglobin

Peroxide-induced oxidative modifications of haem proteins such as myoglobin and haemoglobin can lead to the formation of a covalent bond between the haem and globin. These haem to protein cross-linked forms of myoglobin and haemoglobin are cytotoxic and have been identified in pathological conditions in vivo. An understanding of the mechanism of haem to protein cross-link formation could provide important information on the mechanisms of the oxidative processes that lead to pathological complications associated with the formation of these altered myoglobins and haemoglobins. We have re-examined the mechanism of the formation of haem to protein cross-link to test the previously reported hypothesis that the haem forms a covalent bond to the protein via the tyrosine 103 residue (Catalano, C. E., Choe, Y. S., Ortiz de Montellano, P. R., J. Biol. Chem. 1989, 10534 - 10541). Comparison of native horse myoglobin, recombinant sperm whale myoglobin and Tyr(103) --> Phe sperm whale mutant shows that, contrary to the previously proposed mechanism of haem to protein cross-link formation, the absence of tyrosine 103 has no impact on the formation of haem to protein cross-links. In contrast, we have found that engineered myoglobins that lack the distal histidine residue either cannot generate haem to protein cross-links or show greatly suppressed levels of modified protein. Moreover, addition of a distal histidine to myoglobin from Aplysia limacina, that naturally lacks this histidine, restores the haem protein's capacity to generate haem to protein cross-links. The distal histidine is, therefore, vital for the formation of haem to protein cross-link and we explore this outcome.

DNA–Protein Cross-link Formation in Burkitt Lymphoma Cells Cultured with Benzaldehyde and the Sedative Paraldehyde

Exposure to aldehydes represents potential risks to human and animal health. Cyclic aldehydes such as benzaldehyde, 2-furaldehyde, and paraldehyde were found to induce formation of stable DNA–protein cross-links (DPXs) in cultured human lymphoma cells. A relationship between increased cytotoxicity and DPX formation was observed with each aldehyde. Paraldehyde is a sedative drug used predominately in treatment of ethanol withdrawal. Paraldehyde was the most potent cross-linking aldehyde studied, yet least cytotoxic. Although DPX formation by aliphatic aldehydes is well-known, this study confirms the potential for cyclic aldehydes to cause formation of DPXs in cultured cells at therapeutically relevant doses.

Protein and Low Molecular Mass Thiols as Targets and Inhibitors of Glycation Reactions

Protein glycation has been implicated in the aging process as well as the complications of diabetes (retinopathy, neuropathy, nephropathy, and atherosclerosis). The nitrogen substituents of Lys, Arg, and His residues and the N-terminus of proteins are known to be readily glycated. As the thiol group of Cys is a powerful nucleophile, we hypothesized that Cys residues should also be targets of glycation and that low molecular mass thiols may act as protective agents. In this study the role of thiol glycation, induced by dicarbonyls, in protein cross-link formation and damage prevention is examined. It is shown that incubation of creatine kinase with glyoxal results in protein cross-link formation, with this occurring concurrently with loss of thiol groups, enzyme inactivation, and formation of S-carboxymethylcysteine, a product of glyoxal adduction to Cys residues. Cross-links have also been detected between N-acetylcysteine and the Lys-rich protein histone H1, demonstrating the formation of thiol-glyoxal-amine cross-links. Mass spectrometry has been used to characterize some of these cross-links as 2-(alkylthio)acetamides. A range of low molecular mass thiols have been shown to inhibit dicarbonyl adduction to, and cross-linking of, the thiol-free protein lysozyme, consistent with these thiols being alternative (sacrificial) targets of glycation. Some of these thiols are more efficient modulators of glycation than established glycation inhibitors such as aminoguanidine. These data demonstrate that thiols are facile targets of glycation and that low molecular mass thiols are potent glycation inhibitors. These data may aid the design of therapeutic agents for the treatment of the complications of diabetes.

Structure-Toxicity Analysis of Type-2 Alkenes: In Vitro Neurotoxicity

Acrylamide (ACR) is a conjugated type-2 alkene that produces synaptic toxicity presumably by sulfhydryl adduction. The alpha,beta-unsaturated carbonyl of ACR is a soft electrophile and, therefore, adduction of nucleophilic thiol groups could occur through a conjugate (Michael) addition reaction. To address the mechanism of thiol adduct formation and corresponding neurotoxicological importance, we defined structure-toxicity relationships among a series of conjugated type-2 alkenes (1 microM-10mM), which included acrolein and methylvinyl ketone. Results show that exposure of rat striatal synaptosomes to these chemicals produced parallel, concentration-dependent neurotoxic effects that were correlated to loss of free sulfhydryl groups. Although differences in relative potency were evident, all conjugated analogs tested were equiefficacious with respect to maximal neurotoxicity achieved. In contrast, nonconjugated alkene or aldehyde congeners did not cause synaptosomal dysfunction or sulfhydryl loss. Acrolein and other alpha,beta-unsaturated carbonyls are bifunctional (electrophilic reactivity at the C-1 and C-3 positions) and could produce in vitro neurotoxicity by forming protein cross-links rather than thiol monoadducts. Immunoblot analysis detected slower migrating, presumably derivatized, synaptosomal proteins only at very high acrolein concentrations (>or= 25 mM). Exposure of synaptosomes to high concentrations of ACR (1M), N-ethylmaleimide (10mM), and methyl vinyl ketone (MVK) (100mM) did not alter the gel migration of synaptosomal proteins. Furthermore, hydralazine (1mM), which blocks the formation of protein cross-links, did not affect in vitro acrolein neurotoxicity. Thus, type-2-conjugated alkenes produced synaptosomal toxicity that was linked to a loss of thiol content. This is consistent with our hypothesis that the mechanism of ACR neurotoxicity involves formation of Michael adducts with protein sulfhydryl groups.

MRNA Induction and Cytokine Release of Inflammatory Mediators During In Vitro Exposure of Human Nasal Respiratory Epithelia to Acetaldehyde

Acetaldehyde has been shown to be cytotoxic and carcinogenic to the upper respiratory tract epithelium of rodents following long-term exposure. Most animal studies have concentrated on carcinogenicity and DNA–protein cross-link formation, while less is known about potential dose- and time-dependent induction of aldehyde-induced rhinitis in humans. In this in vitro study, 22 primary cell cultures established from inferior turbinate tissue of healthy individuals were exposed to acetaldehyde concentrations of 50 (German MAK value) or 500 ppm for 4 or 24 h. mRNA expression and protein levels of cytokines and other inflammatory mediators were quantified at the end of the 4- and 24-h exposures. Controls were exposed to synthetic air. Quantitative polymerase chain reaction (Q-PCR) analysis was performed for interleukin (IL)-6, IL-8, IL-1β, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-alpha, GMCSF, Cox-1, and Cox-2. Enzyme-linked immunosorbent assay (ELISA) was performed from culture supernatants for IL-6, IL-8, IL-1β, MCP-1, TNF-alpha, and GMCSF. Significant inductions of IL-1β, TNF-alpha, and Cox-1 and Cox-2 mRNA were observed following exposure to ≥50 ppm acetaldehyde for 4 h. IL-6 and MCP-1 were also induced following a 4-h exposure to 500 ppm acetaldehyde. For all these parameters, effects were significantly stronger at the higher concentration. After 24-h of exposure only Cox-2 remained significantly elevated at 500 ppm but not at 50 ppm, while all other mediators had been downregulated. The obtained data suggest that with exposure to 500 ppm and remarkably also at the level of the occupational exposure limit of 50 ppm, an immediate transient upregulation of inflammatory mediator mRNA is induced, possibly leading to subclinical inflammatory effects.

A Chemical Model for Redox Regulation of Protein Tyrosine Phosphatase 1B (PTP1B) Activity

Growing evidence indicates that endogenously produced hydrogen peroxide acts as a cellular signaling molecule that (among other things) can regulate the activity of some protein phosphatases. Recent X-ray crystallographic studies revealed an unexpected chemical transformation underlying the redox regulation of protein tyrosine phosphatase 1B, in which oxidative inactivation of the enzyme yields an intrastrand protein cross-link between the catalytic cysteine residue and its neighboring amide nitrogen. This work describes a small organic molecule that serves as an effective model for the redox-sensing assembly of functional groups at the active site of PTP1B. Findings obtained using this model system suggest that the oxidative transformation of PTP1B to its "crosslinked" inactive form can proceed directly via oxidation of the active-site cysteine to a sulfenic acid (RSOH). The remarkably facile nature of this protein cross-link-forming reaction, along with the widespread cellular occurrence of protein sulfenic acids generated via oxidation of cysteine residues, suggests that the type of oxidative protein cross-link formation first seen in the context of PTP1B represents a potentially general mechanism for redox "switching" of protein function. Thus, the chemistry characterized here could have broad relevance to both redox-regulated signal transduction and the toxic effects of oxidative stress.

Tissue transglutaminase during mouse central nervous system development: Lack of alternative RNA processing and implications for its role(s) in murine models of neurotrauma and neurodegeneration

Tissue transglutaminase (tTG) is a member of a multigene family principally involved in catalyzing the formation of protein cross-links. Unlike other members of the transglutaminase family, tTG is multifunctional since it also serves as a guanosine triphosphate (GTP) binding protein (Gα h) and participates in cell adhesion. Different isoforms of tTG can be produced by proteolysis or alternative splicing. We find that tTG mRNA is expressed at low levels in the mouse CNS relative to other tissues, and at lower levels in the CNS of mouse in comparison to that of human or rat. tTG mRNA levels are higher in the heart compared to the CNS, for example, and much higher in the liver. Within the CNS, tTG message is lowest in the adult cerebellum and thalamus and highest in the frontal cortex and striatum. In the hippocampus, tTG expression is highest during embryonic development and falls off dramatically after 1 week of life. We did not find alternative splicing of the mouse tTG. At the protein level, the predominant isoform is ∼62 kDa. In summary, tTG, an important factor in neuronal survival, is expressed at low levels in the mouse CNS and, unlike rat and human tTG, does not appear to be regulated by alternative splicing. These findings have implications for analyses of rodent tTG expression in human neurodegenerative and neurotrauma models where alternative processing may be an attractive pathogenetic mechanism. They further impact on drug discovery paradigms, where modulation of activity may have therapeutic value.

Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine.

Transglutaminase 2 (TGase 2) is one of a family of enzymes that catalyze protein modification through the incorporation of polyamines into substrates or the formation of protein crosslinks. However, the physiological roles of TGase 2 are largely unknown. To elucidate the functions of TGase 2, we have searched for its interacting proteins. Here we show that TGase 2 interacts with E7 oncoprotein of human papillomavirus type 18 (HPV18) in vitro and in vivo. TGase 2 incorporates polyamines into a conserved glutamine residue in the zinc-binding domain of HPV18 E7 protein. This modification mediates the inhibition of E7's Rb binding ability. In contrast, TGase 2 does not affect HPV16 E7, due to absence of a glutamine residue at this polyamination site. Using E7 mutants, we demonstrate that TGase 2-dependent inhibition of HPV E7 function correlates with the presence of the polyamination site. Our results indicate that TGase 2 is an important cellular interfering factor and define a novel host-virus interaction, suggesting that the inability of TGase 2 to inactivate HPV16 E7 could explain the high prevalence of HPV16 in cervical cancer.

Plant phenolics as cross-linkers of gelatin gels and gelatin-based coacervates for use as food ingredients

Polyphenols are known to react under oxidizing conditions with side chain amino groups of peptides, leading to formation of cross-links in proteins. Plant-derived phenolic acids and flavonoids were used to prepare cross-linked gelatin gels in bulk and cross-linked gelatin–pectin coacervates in the form of microparticles for use as food ingredients. Gels cross-linked by these materials had greater mechanical strength, reduced swelling, and fewer free amino groups. Dynamic light scattering analyses showed that such cross-linking results in denser polymeric networks and prevents extension of the peptide chains is when the pH is moved away from the isoelectric point. Coacervated gelatin–pectin microparticles when cross-linked became more lipophilic, and were stable at temperatures up to 200 °C, in contrast to un-cross-linked particles that coalesce and/or disintegrate on heating. These properties of cross-linked gelatin gels and gelatin-based coacervates have applications for the development of novel food ingredients.

Does tissue transglutaminase play a role in Huntington's disease?

Tissue transglutaminase (tTG) likely plays a role in numerous processes in the nervous system. tTG posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines (Glns). This reaction results in the incorporation of polyamines into substrate proteins or the formation of protein crosslinks, modifications that likely have significant effects on neural function. Huntington's disease is a genetic disorder caused by an expansion of the polyglutamine domain in the huntingtin protein. Because a polypeptide bound Gln is the determining factor for a tTG substrate, and mutant huntingtin aggregates have been found in Huntington's disease brain, it has been hypothesized that tTG may contribute to the pathogenesis of Huntington's disease. In vitro, polyglutamine constructs and huntingtin are substrates of tTG. Further, the levels of tTG and TG activity are elevated in Huntington's disease brain and immunohistochemical studies have demonstrated that there is an increase in tTG reactivity in affected neurons in Huntington's disease. These findings suggest that tTG may play a role in Huntington's disease. However in situ, neither wild type nor mutant huntingtin is modified by tTG. Further, immunocytochemical analysis revealed that tTG is totally excluded from the huntingtin aggregates, and modulation of the expression level of tTG had no effect on the frequency of the aggregates in the cells. Therefore, tTG is not required for the formation of huntingtin aggregates, and likely does not play a role in this process in Huntington's disease brain. However, tTG interacts with truncated huntingtin, and selectively polyaminates proteins that are associated with mutant truncated huntingtin. Given the fact that the levels of polyamines in cells is in the millimolar range and the crosslinking and polyaminating reactions catalyzed by tTG are competing reactions, intracellularly polyamination is likely to be the predominant reaction. Polyamination of proteins is likely to effect their function, and therefore it can be hypothesized that tTG may play a role in the pathogenesis of Huntington's disease by modifying specific proteins and altering their function and/or localization. Further research is required to define the specific role of tTG in Huntington's disease.

Expression of advanced glycation end products and their cellular receptor RAGE in diabetic nephropathy and nondiabetic renal disease.

Advanced glycation end products (AGE) contribute to diabetic tissue injury by two major mechanisms, i.e., the alteration of extracellular matrix architecture through nonenzymatic glycation, with formation of protein crosslinks, and the modulation of cellular functions through interactions with specific cell surface receptors, the best characterized of which is the receptor for AGE (RAGE). Recent evidence suggests that the AGE-RAGE interaction may also be promoted by inflammatory processes and oxidative cellular injury. To characterize the distributions of AGE and RAGE in diabetic kidneys and to determine their specificity for diabetic nephropathy, an immunohistochemical analysis of renal biopsies from patients with diabetic nephropathy (n = 26), hypertensive nephrosclerosis (n = 7), idiopathic focal segmental glomerulosclerosis (n = 11), focal sclerosis secondary to obesity (n = 7), and lupus nephritis (n = 11) and from normal control subjects (n = 2) was performed, using affinity-purified antibodies raised to RAGE and two subclasses of AGE, i.e., N(epsilon)-(carboxymethyl)-lysine (CML) and pentosidine (PENT). AGE were detected equally in diffuse and nodular diabetic nephropathy. CML was the major AGE detected in diabetic mesangium (96%), glomerular basement membranes (GBM) (42%), tubular basement membranes (85%), and vessel walls (96%). In diabetic nephropathy, PENT was preferentially located in interstitial collagen (90%) and was less consistently observed in vessel walls (54%), mesangium (77%), GBM (4%), and tubular basement membranes (31%). RAGE was expressed on normal podocytes and was upregulated in diabetic nephropathy. The restriction of RAGE mRNA expression to glomeruli was confirmed by reverse transcription-PCR analysis of microdissected renal tissue compartments. The extent of mesangial and GBM immunoreactivity for CML, but not PENT, was correlated with the severity of diabetic glomerulosclerosis, as assessed pathologically. CML and PENT were also identified in areas of glomerulosclerosis and arteriosclerosis in idiopathic and secondary focal segmental glomerulosclerosis, hypertensive nephrosclerosis, and lupus nephritis. In active lupus nephritis, CML and PENT were detected in the proliferative glomerular tufts and crescents. In conclusion, CML is a major AGE in renal basement membranes in diabetic nephropathy, and its accumulation involves upregulation of RAGE on podocytes. AGE are also accumulated in acute inflammatory glomerulonephritis secondary to systemic lupus erythematosus, possibly via enzymatic oxidation of glomerular matrix proteins.

Inhibition of angiogenesis on glycated collagen lattices.

Advanced glycation endproduct (AGE) accumulation in extracellular matrix proteins has been demonstrated in diabetic patients with a significant correlation with the severity of diabetic complications. AGE accumulation induces matrix protein cross-link formation, resulting in an increased stiffness of matrix fibres and the reduction of the susceptibility of matrix proteins to proteolytic degradation. We examined whether glycation-induced collagen cross-linking may affect vascular endothelial cell behaviours such as invasion, proliferation and differentiation, using the in vitro angiogenesis model of capillary-like structure formation in three-dimensional matrices of collagen type I. Endothelial cells cultured on collagen gel with angiogenic factors (the combination of fibroblast growth factor-2 and vascular endothelial growth factor) invaded the underlying collagen matrix, and organized capillary-like cord structures in the gel. We found that endothelial cell invasion into glycated collagen gel was significantly attenuated without any effect on proteinase activity including cell-associated plasminogen activator and matrix metalloproteinase in the conditioned medium. In addition, subsequent capillary-like cord formation was also inhibited in glycated collagen gel. In contrast, endothelial cell proliferation was enhanced on glycated collagen gel with or without angiogenic factors compared with control collagen gel. These results suggest that the structural alterations of extracellular matrix proteins through the glycation-induced cross-link formation affect the interaction between endothelial cell and extracellular matrix, resulting in the impairment of an adequate neovascularization in diabetic patients.

Advanced Maillard reaction end products are associated with Alzheimer disease pathology.

During aging long-lived proteins accumulate specific post-translational modifications. One family of modifications, termed Maillard reaction products, are initiated by the condensation between amino groups of proteins and reducing sugars. Protein modification by the Maillard reaction is associated with crosslink formation, decreased protein solubility, and increased protease resistance. Here, we present evidence that the characteristic pathological structures associated with Alzheimer disease contain modifications typical of advanced Maillard reaction end products. Specifically, antibodies against two Maillard end products, pyrraline and pentosidine, immunocytochemically label neurofibrillary tangles and senile plaques in brain tissue from patients with Alzheimer disease. In contrast, little or no staining is observed in apparently healthy neurons of the same brain. The Maillard-reaction-related modifications described herein could account for the biochemical and insolubility properties of the lesions of Alzheimer disease through the formation of protein crosslinks.

Cellular accumulation and DNA damage induced by liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum(II) in LoVo and LoVo/PDD cells

Liposomal cis-bis-neodecanato-trans-R,R-1,2-diaminocyclohexaneplatinum (11) (L-NDDP) is a liposome-entrapped platinum complex that has shown partial lack of cross-resistance with cisplatin in human colon carcinoma LoVo cells. We studied the drug accumulation and DNA damage induced by L-NDDP and cisplatin in LoVo and LoVo/PDD cells. Our results indicate that the accumulation of L-NDDP in LoVo cells is several-fold higher than that of cisplatin; that the accumulation of L-NDDP is similar in both cell lines, whereas that of cisplatin is reduced by 2- to 3-fold in LoVo/PDD cells; and that the transmembrane transport of cisplatin is highly dependent on temperature while that of L-NDDP is not. We also found that the cytotoxicity of both agents correlates with the extent of DNA-protein cross-link formation, and that DNA interstrand cross-linking does not appear to play a role in the cytotoxicity of L-NDDP, whereas it correlates with cisplatin cytotoxicity.

Induction of DNA damage in gamma-irradiated nuclei stripped of nuclear protein classes: differential modulation of double-strand break and DNA-protein crosslink formation.

The influence of chromatin proteins on the induction of DNA double-strand breaks (dsb) and DNA-protein crosslinks (dpc) by gamma-radiation was investigated. Low molecular weight non-histone proteins and classes of histones were extracted with increasing concentrations of NaCl, whereas nuclear matrix proteins were not extractable even by 2.0 M NaCl. The yield of dsb increased with progressive removal of proteins from chromatin. Whilst removal of low molecular weight non-histone proteins and histone H1 resulted in small increases in the production of dsb, removal of histones H2A/H2B, all histones, or all proteins led to 18.4, 46.4 and 55.5-fold increases in the yield of dsb, respectively, relative to irradiated cells. Therefore, both histones and non-histone proteins contribute to the radioprotection of DNA, core histones being the major radio-protectors. In contrast, depletion of chromatin proteins caused little or no effect on the induction of dpc until the chromatin was extracted with > or = 1.4 M NaCl. However, our studies indicated no direct, quantitative correlation between the removal of histones and the induction of dpc. The data support our previous conclusion that nuclear matrix protein rather than the majority of the histones are the predominant substrates for dpc production, although the involvement of a subset of tightly bound histones (H3 and H4) has not been excluded. This finding demonstrates that chromatin proteins can differentially modify the yield of two types of radiation-induced DNA lesions.

Differences in the post-translational modification of proteins by polyamines between weakly and highly metastatic B16 melanoma cells.

The identification of (gamma-glutamyl)polyamines in proteolytic digest of proteins from the cytosolic and particulate fractions of B16-F10 and B16-F10Lr6 cell lines, originating from a spontaneous tumor in C57BL/6 mice, indicates that polyamines are incorporated into melanoma cell proteins by transglutaminases (TGases-EC 2.3.2.13). The levels of spermidine-derived protein cross-links were found to be inversely related with the metastatic potential of the 2 melanoma lines. Characterization of TGase activity in the 2 tumor cell lines showed 3 types of enzyme. The soluble cellular TGase activity (TGase C) was higher, and increased more, during the growth of the least metastasizing clone B16-F10Lr6 than in the B16-F10 line, which is the most metastasizing. Consistently, N1,N8-bis(gamma-glutamyl) spermidine, which is responsible for protein cross-link formation, was present in greater amount in B16-F10Lr6 cells. The enhancement by theophylline of soluble-TGase activity and spermidine-dependent protein cross-links of B16-F10 cells reduced, with linear dose dependence, the ability of these cells to penetrate through human fibronectin-coated membrane in an in vitro assay of invasiveness. Our data confirm and extend earlier observations indicating that the propensity of a tumor to metastasize can be indirectly related to intracellular levels of TGase activity, and provide the basis for some speculation concerning the role of polyamines as modifiers of murine melanoma cell proteins in metastasis.

Transglutaminase modification of rhodopsin in retinal rod outer segment disk membranes

Rhodopsin in rod outer segment disk membranes was enzymatically modified by erythrocyte transglutaminase, which linked small primary amines to glutamine residues. In order to avoid formation of protein crosslinks, rhodopsin was first reductively methylated to modify its lysines. From 1.9 to 2.5 mol of putrescine, ethanolamine, or dinitrophenylcadaverine were incorporated into rhodopsin by transglutaminase during 16 h reaction time. A maximum of 3.5 mol of [ 14C]putrescine was incorporated per mole of rhodopsin during 48 h. Essentially all of the rhodopsin sequence containing the putrescine could be removed by limited proteolysis of the membranes by thermolysin. Glutamine residues in positions 236, 237, 238, and 344 were modified to approximately equal extents, as determined by isolation of the cyanogen bromide peptides of modified rhodopsin followed by further subdigestion of the peptides. The modified glutamine residues are located in the helix V–VI (or F1–F2) connecting loop and in the carboxyl-terminal region of rhodopsin.

Formation of dityrosine cross-links in proteins by oxidation of tyrosine residues

1. 1.|Enzymic oxidation of proteins with peroxidase and hydrogenperoxide at a basic pH value leads to an oxidative phenolic coupling of adjacent tyrosine residues forming cross-linked proteins. 2. 2.|Dityrosine (3,3′-bityrosine) was identified as the cross-link in oxidised proteins by thin-layer chromatography, amino acid analysis and fluorescence measurements. 3. 3.|Gel filtration experiments with oxidised insulin showed that the cross-linkage is predominantly intermolecular. 4. 4.|In tetranitromethane treated proteins, dityrosine could be identified after hydrolysis.