Degradation Of Cystine Research Articles

Contribution of Amino Acid Metabolism to Hematologic Malignancies

Tumor cells rewire metabolic pathways to meet the high metabolic demands of proliferation, frequently developing auxotrophy to specific amino acid(s) (AAs) required to satisfy protein biosynthesis. Thus specific metabolic inhibitors or AA-depleting enzymes have been developed and tested as cancer therapeutics. For example, depletion of asparagine by bacterial L-asparaginase (ASNase) has proven efficacious against hematologic malignancies, especially leukemia and lymphoma, by starving tumors lacking asparagine synthetase (ASNS). We and others have reported that the glutaminase (GLS) activity of ASNase is required for anticancer activity against ASNS-positive leukemia cell types in vitro.1 In vivo, we have found that durable response to ASNase in pre-clinical models of leukemia also requires glutaminase activity, even against ASNS-negative leukemia models; a glutaminase-deficient mutant of ASNase yielded subsequent leukemia recurrence. We speculate that the underlying anti-leukemia mechanism mediated by ASNase glutaminase activity involves a deeper depletion of asparagine within the tumor microenvironment, since ASNS in nearby cells (adipocytes, mesenchymal stromal cells, etc.) can use glutamine as a precursor for asparagine synthesis. Nevertheless, since L-glutamine depletion is thought to cause the significant side effects of ASNase, enzyme variants with reduced glutaminase coactivity are being developed and tested. Another viable therapeutic strategy involving glutamine starvation via GLS inhibitor has shown significant pre-clinical activity in acute myeloid leukemia (AML) and multiple myeloma (MM) models; this approach is synergistic with hypomethylating agents and BCL2 inhibitors in AML, and with proteasome inhibitors in MM. Recent findings highlight the switch to glutamine metabolism as a metabolic dependency of tyrosine kinase-driven AML, and targeting GLS in conjunction with tyrosine kinase inhibition has been proposed.2 Targeting arginine metabolism has been shown to be another viable therapeutic strategy. Arginine (ARG) depletion using pegylated arginine deaminase (ADI-PEG 20) or pegylated arginase (PEG-ARGase), the 2 critical enzymes of the ARG metabolism/urea cycle, reduced leukemia tumor burden in AML models characterized by low arginosuccinate synthetase (ASS) and high uptake of ARG. However, recently reported Phase I/II clinical trials of recombinant PEG-arginase and of ADI-PEG 20 showed minimal efficacy in relapsed/refractory AML and in solid tumors despite efficient depletion of arginine and low ASS1 expression in tumors, indicating that depletion of arginine alone is insufficient for clinical activity. As a final example of AA metabolic pathways targeted in the treatment of hematologic malignancies, exogenous L-cysteine is required for the synthesis of glutathione for antioxidant cellular defense. In pre-clinical studies, multiple malignancy subtypes were sensitive to cysteine and cystine degradation by an engineered human cyst(e)inase enzyme, including AML, acute lymphocytic leukemia, poor-risk chronic lymphocytic leukemia (CLL), and MM.3 In all therapeutic strategies targeting AA metabolism, the tumor microenvironment may contribute to resistance. For example, bone marrow stromal cells efficiently import cystine, convert it to cysteine, and transport it to CLL cells, facilitating leukemia chemoresistance. Mesenchymal stromal cells and bone marrow adipocytes secrete asparagine and glutamine, respectively, and protect leukemia cells from ASNase cytotoxicity. Recent insights into the immune tumor microenvironment highlight interplay between tumor, AAs, and immune cell functions. Some AAs, such as arginine and glutamine, are essential nutrients for immune cell proliferation and metabolism; excessive tumor consumption of glutamine, or secretion of arginase by myeloid-derived suppressor cells or AML blasts, may deprive immune cells, impair T cell proliferation, and induce immunosuppressive phenotypes. GLS inhibitors that block glutamine consumption and arginase inhibitors that increase plasma arginine, increase availability of their respective target nutrients for immune cells and are, therefore, being explored in ongoing clinical trials as monotherapies and in combination with anti-PD1 blockade.Chan WK, Lorenzi PL, Anishkin A, et al. The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells. Blood. 2014;123:3596-3606.Gallipoli P, Giotopoulos G, Tzelepis K, et al. Glutaminolysis is a metabolic dependency in FLT3(ITD) acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition. Blood. 2018;131:1639-1653.Zhang W, Trachootham D, Liu J, et al. Stromal control of cystine metabolism promotes cancer cell survival in chronic lymphocytic leukaemia. Nat Cell Biol. 2012;14:276-286. DisclosuresKonopleva:Stemline Therapeutics: Research Funding. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties.

Effects of protein sources on concentrations of hydrogen sulphide in the rumen headspace gas of dairy cows

Two Latin square design experiments investigated the relationship between hydrogen sulphide concentration in the rumen headspace gas of dairy cows and the early stages of protein degradation in the rumen. In Expt 1, three protein sources differing in rumen N (nitrogen) degradability (maize gluten feed (MGF); sunflower meal (SFM); and soyabean meal (SBM)) were used, whereas in Expt 2 four different batches of the same feed (MGF) differing in colour (CIE L*, a*, b* (CIELAB) scale) were used. After allowing the concentration of hydrogen sulphide in rumen gas to decline close to zero, a fixed amount of protein sources was offered to cows and the concentrations of hydrogen sulphide were recorded in rumen headspace gas at 30-min intervals. In Expt 1, the concentration of hydrogen sulphide showed considerable variation between protein sources, with MGF having the highest concentration followed by SFM and SBM resulting in very low concentrations. The N wash losses (zero time measurements with nylon bags) ranked the feeds in the same way, from MGF (highest; 61%) to SBM (lowest; 26%). There were marked differences in the degradation of cystine and methionine between protein sources, although the degradation of cystine was always higher than for methionine. MGF (Expt 2) led to increased concentrations of hydrogen sulphide, with peak concentrations achieved between 1 and 2 h after feeding. The concentrations of hydrogen sulphide were higher for MGF1, intermediate for MGF2 and lower for MGF3 and MGF4, agreeing with colour scale. Differences in the early stages of dietary sulphur degradation corresponded with differences in hydrogen sulphide concentrations in rumen gas. The results suggest that hydrogen sulphide concentrations in the rumen headspace gas could be useful to evaluate nutritional parameters not measured by the in sacco technique, contributing to a better understanding of the response of dairy cows to different protein supplements.

The Partial Protection of Wool Against Photo-oxidation by Application of Reactive Dyes*

Wool dyed under different conditions was irradiated in a Xenotest apparatus in the presence of moist air. The photo–oxidative changes in the functional groups of the wool were investigated using an amino acid analyser. Undyed control samples were more decomposed than the dyed samples. In the presence of the dye, degradation of cystine, which is the amino acid essential for wool stability, was considerably less than in native wool. A smaller protective effect of the dye was observed on the wool side–chains lysine, histidine and tyrosine. With methionine, however, degradation was higher. Furthermore, numerous other amino–acid side–chains were decomposed in both dyed and blank samples, producing many degradation products. Aminoadipic acid was one of the oxidation products of lysine. The covalent bond between fibre and dye was stable under the irradiation conditions used.

Effects of Anaerobic Atmosphere on the Metabolism of Sulfur Volatile Compounds Produced by Broccoli

Objectionable off-odors are produced by broccoli (Brassica oleracea, L.) when it is held under anaerobic conditions. These off-odors were attributed to sulfur volatile compounds mainly methanethiol (MT) and hydrogen sulfide. The present study was undertaken to investigate the effects of anaerobic conditions on the metabolism and emission of sulfur volatiles by broccoli. Inhibition assays using aminooxyacetic acid (AOA)—a potential inhibitor of pyridoxal-phosphate-dependent enzymes-confirmed the enzymatic origin of these volatiles. However, anaerobic atmosphere had no inducible effect on the enzymes cystine lyase, cysteine desulfhydrases and S-alkylcysteine lyase. These pyridoxal-phosphate-dependent enzymes thought to catalyze the respective degradation of cystine, cysteine and S-methyl-L-cysteine to sulfur volatiles showed no significant activity increase. Storage of sterile broccoli seedlings under anaerobic atmosphere resulted in an important increase of the content of sulfur amino acids that corresponded to an increased emission of sulfur volatiles. Cysteine and methionine content increased particularly at 24 hours and decreased later. Whereas, S-methyl-L-cysteine content increase was more obvious after 48 hours. The results suggest a possible involvement of the pathways for synthesis and breakdown of sulfur amino acids via methionine.

Crosslinking nucleophilic dyes on wool

Nucleophilic amino-alkyl dyes have been synthesised by the simple expedient of reacting existing reactive dyes with ethylene diamine. These dyes when applied to wool behave as acid dyes, although there is a small amount of covalent bonding to the fibre, presumably through Michael addition at dehydroalanine residues formed from cystine degradation. If substantive crosslinking agents could be applied then it might be expected that excellent dye-fibre covalent bonding could be achieved to produce dyeings with very high wet fastness properties. The crosslinking agent might be added either before, during or after the amino-alkyl dye application; the latter procedure should, in principle, give the most level dyeings. Crosslinking agents evaluated include hexamine, triacroylamino-hexahydro- s-triazine, methylene-bisacrylamide and 2-chloro-4.6-di(aminobenzene-4′-sul-phatoethylsulphone)- s-triazine. Hexamine and methylene-bisacrylamide are available commercially but the other agents were synthesised. Results obtained are very promising and offer potential wool dyeing methods which have better levelness than existing reactive dye systems.

Maillard Reaction Products and Lysinoalanine

Heat processing is essential for the preservation of milk-based infant formulas. Heating, however, induces a number of chemical changes during which lysine in the milk proteins reacts with reducing sugars to form Maillard reaction products (MRPs) and also reacts with the dehydroalanine resulting from cystine degradation to form lysinoalanine (LAL). Both products have been reported to induce histological changes in the straight portion of the proximal tubule in the rat kidney. This pilot study was made to investigate the urinary excretion by healthy preterm babies of MRPs and LAL contained in infant formula and to determine their influence on kidney function. Twelve healthy male preterm babies were first fed for 10 days with pooled human milk and then for 5 days with each of two experimental premature infant formulas in a cross-over design. The infant formulas were sterilized either by ultra-high temperature (UHT) treatment or by a conventional retort process to give products with low and high levels of MRPs and LAL, respectively. In total, some 15.6% of the initial lysine had been modified in the in-can-sterilized product, compared to 6.2% in the UHT product. Urinary excretion of MRP lactulosyllysine ranged from 1.3 to 3.9% of the ingested amount, whereas that of LAL ranged from 6.2 to 9.3%. The higher level of MRPs and LAL in the formulas compared to breast milk had no influence on creatinine clearance or electrolyte excretion. There was no evidence of tubular damage as determined by the urinary excretion of four kidney-derived enzymes. Feeding of formula, however, did result in a general increase in urinary microprotein levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Maillard Reaction Products and Lysinoalanine

SummaryHeat processing is essential for the preservation of milk‐based infant formulas. Heating, however, induces a number of chemical changes during which lysine in the milk proteins reacts with reducing sugars to form Maillard reaction products (MRPs) and also reacts with the dehydroalanine resulting from cystine degradation to form lysinoalanine (LAL). Both products have been reported to induce histological changes in the straight portion of the proximal tubule in the rat kidney. This pilot study was made to investigate the urinary excretion by healthy preterm babies of MRPs and LAL contained in infant formula and to determine their influence on kidney function. Twelve healthy male preterm babies were first fed for 10 days with pooled human milk and then for 5 days with each of two experimental premature infant formulas in a cross‐over design. The infant formulas were sterilized either by ultra‐high temperature (UHT) treatment or by a conventional retort process to give products with low and high levels of MRPs and LAL, respectively. In total, some 15.6% of the initial lysine had been modified in the in‐can‐sterilized product, compared to 6.2% in the UHT product. Urinary excretion of MRP lactulosyllysine ranged from 1.3 to 3.9% of the ingested amount, whereas that of LAL ranged from 6.2 to 9.3%. The higher level of MRPs and LAL in the formulas compared to breast milk had no influence on creatinine clearance or electrolyte excretion. There was no evidence of tubular damage as determined by the urinary excretion of four kidney‐derived enzymes. Feeding of formula, however, did result in a general increase in urinary microprotein levels. This did not seem to be a reflection of tubular impairment and was probably due to a transient and normal adaptation to the higher protein content of the formulas, although an effect of MRPs and LAL could not be ruled out.

Investigation of the Alkaline Modification of Wool Using New Analytical Methods for the Determination of Degradation

In order to meet the requirements for brilliant colours and high wet fastness, the cotton component of wool/cotton blends is often dyed with reactive dyes under strongly alkaline conditions. A modified enzymatic method has been used to determine dehydroalanine residues formed in wool during commercial dyeing treatments. Reaction mechanisms of cystine degradation have been studied on the basis of the amounts of dehydroalanine present. Hydrogen sulphide could be determined quantitatively in wool and in dyebaths using an improved method that minimises further cystine degradation and hydrogen sulphide oxidation.

Cystine catabolism in mycelia of Microsporum gypseum, a dermatophytic fungus.

The fate of 35S label was studied during cystine degradation by mycelia of the dermatophytic fungus Microsporum gypseum. Excess free cystine in the medium was readily taken up and its sulfur moiety excreted as inorganic sulfate and sulfite. At intervals after 3-60 min of incubation with 35S cystine the products of cystine catabolism were extracted from the mycelia by boiling water and separated by thin layer chromatography and electrophoresis. A total of 10 sulfur-containing compounds were identified, and their relative radioactivity was assessed. After 3 min the mycelia contained, in addition to cystine, labeled cysteine and particularly cysteine sulfinic acid which was accompanied by a smaller amount of cysteic acid. Later on, oxidized and reduced glutathione, inorganic sulfate and taurine appeared consecutively. In all extracts, small amounts of labeled S-sulfocysteine were found, not, however, sulfite. The results suggest that the intermediates of cysteine degradation in the fungal mycelia are cysteine, cysteine sulfinate, unstable sulfinylpyruvate, sulfite and sulfate, i.e., that the catabolic pattern is similar to that of higher organisms. The formation and the role of S-sulfocysteine, cysteic acid, and of taurine is not yet completely understood, although certainly autoxidative processes are involved in the formation of the latter two compounds, and sulfitolysis in that of the former compound.

The effect of varying autoclaving conditions and added sodium hydroxide on amino acid content and nitrogen characteristics of feather meal

AbstractThe effects upon amino acid content and nitrogen characteristics of feather meal subjected to varying lengths of processing time (30 to 70 min) moisture content (50 to 70%) and added NaOH (0.2 to 0.6%) were studied by multiple regression techniques. All test products were autoclaved at constant pressure of 345 kPa. The results obtained showed that prolonged processing periods and increased concentrations of NaOH had a substantial effect on the amino acid contents and in particular on the essential ones. There was a marked degradation of cystine, and smaller losses of lysine and methionine. Most of the amino acid contents were lower in the meals with a higher moisture content. In vitro protein digestibility and solubility in alkali and acid, were generally increased by prolonged processing conditions.It was concluded that the inverse relationship between amino acid content and nitrogen characteristics should be taken into account for feather meal evaluation. Higher values for in vitro protein digestibility and solubility were considered to be favourable unless other structural modifications to the protein affect its nutritional value.

Amino acid content and protein solubility of feather meal as affected by different processing conditions.

Feathers of broiler chickens were autoclaved for 30 to 70 min and feather moisture content was varied from 50 to 70% with no addition, addition of 0.2 to 0.6% sodium hydroxide or addition of 0.2 to 0.6% proteolytic enzyme. During processing there was marked degradation of cystine and smaller losses of the limiting amino acids lysine, methionine and histidine. Most of the lost cystine seemed to be converted to lanthionine. Digestibility (in pepsin-HCl solution) and solubility (in alkali or acid solvents) of feather meal protein were increased as a result of processing. (Abstract retrieved from CAB Abstracts by CABI’s permission)

Volatile components of the thermal degradation of cystine in water

Volatile components of the thermal degradation of cystine in water

Degradative addition reactions in relation to protein‐bound cystine

AbstractA study has been made of the degradation of protein‐bound cystine, in various proteins, when degraded by alkali in the presence of aliphatic amines. This shows that the structure of amine has a significant effect on the rate of degradation of the cystine. The products of degradation of cystine which resulted from the intermediate, dehydroalanine, have been determined quantitatively at different temperatures of treatment. Attempts are made to correlate the amounts of products with the nature of the amines used and the hydrophobicity of the proteins.

Cystine lyase: β-cystathionase from turnip roots

Cystine lyase (EC 4.4.1.-) was purified 277-fold by a combination of ammonium sulfate precipitation, chromatography on calcium phosphate and DEAE-cellulose with a 6% recovery. The MW as measured by gel filtration on Biogel p-300 was ca 150 000. The enzyme catalysed the pyridoxal phosphate-dependent degradation of cystine to pyruvate, ammonia and cysteine persulfide. Cysteine persulfide normally degraded spontaneously to elemental sulfur and cysteine, that further reacted to yield cystine and H 2S. Pyridoxal phosphate stabilized the enzyme. The K m value for cystine was 0.94 mM. The enzyme was insensitive to thiol reagents but was inhibited by some thiols (which may have reduced the cystine). Cystine lyase degraded many compounds having the L-α-amino propionic acid group with a thioether or disulfide linkage attached to the β-carbon but was inactive towards D-configuration at the α-carbon or L-homocystine. The cystine lyase was also a β-cystathionase as indicated by (1) a constant ratio of β-cystathionase activity to cystine lyase activity throughout a 277-fold purification, (2) the inhibition of cystine lyase activity by cystathionine and inhibition of β-cystathionase activity by cystine and (3) similarity in sensitivity to heat, cyanide and hydroxylamine. Using DL-cystathionine as substrate, the K m value was 4 mM.

Cystine-alkali reactions in relation to protein crosslinking.

The formation of lysinoalanine, lanthionine and β-aminoalanine in proteins, through the alkaline degradation of cystine residues to dehydroalanyl residues, is discussed. The formation of these amino-acids, during the dry heating of proteins, is also reported. It is shown that, during alkaline degradation of protein-bound cystine, amino or thiol-containing compounds present in the alkaline liquor can compete with protein-bound amino and thiol groups for addition to the dehydroalanyl residues formed as intermediates in the reaction. Results showing the relative importance of such competitive reactions are given. Evidence given indicates the one protein-bound cystine residue can, on alkaline degradation, yield two dehydroalanyl residues. The extent of addition of a series of alkylamines, to the transient dehydroalanyl residues, is shown to be dependent on the structure of the pendant alkyl group. Addition is governed by two factors, activity of the addendum, and the positioning of possible addenda in relation to the protein-bound dehydroalanyl residues, when the latter are formed. Dyeing experiments with alkylamine-treated wools show that the pendant alkyl groups influence dyeability. The results indicate that hydrophobic interactions in the modified protein have as great an effect on dyeability as do covalent crosslinks and ionic groups. Dye uptake is determined by the shape and size of the pendant alkyl groups introduced during the protein modification, rather then the enhanced basicity caused by introduction of the basic sidechains. The possibility of introducing hydrophobic sidechains into proteins, as amide derivatives of protein-bound lysine residues, by simple dry heating, is speculated upon, in relation to dry-heating of a fat-containing protein.KeywordsSilk FibroinWool FibreAlkaline DegradationWool KeratinCystine ResidueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Nährwertverluste von Proteinen in Fleischprodukten als Folge der thermischen Verarbeitung

In certain cases, the proteins in meat products may be damaged during thermal processing to such an extent that their nutritional value is reduced. This might be caused above all by a loss in the availability of the essential and semi-essential amino-acids. Cooking of the meat produces no marked change in the availability of the amino acids. More marked changes are observed at temperatures needed for sterilization. The availability of cystine is in most cases affected by the length and temperature of sterilization, which is due to a reduction of its digestibility and to its degradation. The extent of the cystine degradation follows from the difference from the average reduction of the availability of the other amino acids. Since the sulphur-containing amino acids in meat proteins are limiting ones, this loss results in a reduction of the nutritional value of the proteins. Apart from cystine, lysine may also become less available if greater amounts of polysaccharides are present. The reduction of the availability can be so marked that lysine may be considered to be a limiting amino acid. In these cases, lysine is essential for the nutritional value of the proteins in such products.

The Covalent Reactions of Primary Amino Side–chains and Disulphide Bonds in Wool Keratin

The reactions with alkali of the disulphide bonds and the primary amino side–chains in wool have been investigated. Formation of dehydroalamine has been postulated and it has been conclusively identified in an alkali–treated, cystine–containing polypeptide. The mechanism of degradation of cystine in such a peptide has been further investigated and it has been shown that cysteine residues, produced from cystine during degradation, are also capable of degrading further to dehydroalanyl residues. The dehydroalanyl residues so produced are capable of reacting not only with other residues to form LAN and LAL, but also with other species present in the alkaline medium. The influence of amines on the cold setting of wool fibres with thioglycollates and on the dyeability of wool has been studied. Mechanisms are postulated to explain these effects.

2—THE COMPETITIVE ADDITION REACTION OF DEHYDROALANINE RESIDUES FORMED DURING THE ALKALINE DEGRADATION OF WOOL CYSTINE

An investigation is described in which the effect of a thiol-reducing agent, thioglycollic acid, on wool in borate and ammonia solutions was ascertained in relation to the competition of thiols and amines for combination with dehydroalanine residues. Thioglycollate was found to decrease the amounts of products formed by amino-group addition to dehydroalanine residues at pH 12 and to eliminate them completely at pH 10. It is also shown that, at certain concentrations of thioglycollate, the degradation of cystine to dehydroalanine residues is greatly increased to give maximum lanthionine formation, but above this critical concentration thioglycollic acid competes successfully with the thiol group of cysteine to give S-carboxymethylcysteine rather than lanthionine. Differences between alkaline solutions of borates and ammonia with thioglycollate present are ascribed to the differences in reduction of cystine to cysteine; the blocking of free thiol groups is used to observe any difference in the amount of sul...

An investigation of the mechanism of alkaline degradation of cystine in intact protein

The degradative effect of a series of alkyl amines has been determined on the cystine residues in keratin. These have all been found to give rise to large amounts of new β- N-alkyl amino acid residues in the fibre and a relationship between length of alkyl chain and amount of new amino acid found has been shown. These findings can only be rationalised in terms of a modified mechanism for the alkaline degradation of cystine in the protein material, and a mechanism in agreement with the experimental findings is postulated.

The photochemical degradation of cystine in aqueous solutions in the presence of nitrogen.

Abstract Ultraviolet irradiation of cystine in aqueous solution at pH 1 and pH 10 in the presence of nitrogen has been investigated. The products of degradation are far fewer than those observed in the presence of air. Despite the exlusion of oxygen, one sulphur-oxygen compound (alanine 3-sulphinic acid) has been identified and is presumed to be formed by hydrolytic fission and disproportionation. The major decomposition products in both acid and alkaline conditions are cystenine, alanine, lathionine, ammonia, sulphur and hydrogen sulphide. Lanthionine is only observed under acidic conditions.