Amino Lysine Research Articles

SUMO proteases as potential targets for cancer therapy.

Sumoylation is one of the post-translational modifications of proteins, responsible for the regulation of many cellular processes, such as DNA replication and repair, transcription, signal transduction and nuclear transport. During sumoylation, SUMO proteins are covalently attached to the ε-amino group of lysine in target proteins via an enzymatic cascade that requires the sequential action of E1, E2 and E3 enzymes. An important aspect of sumoylation is its reversibility, which involves SUMO-specific proteases called SENPs. SENPs (sentrin/SUMO-specific proteases) catalyze the deconjugation of SUMO proteins using their isopeptidase activity. These enzymes participate through hydrolase activity in the reaction of SUMO protein maturation, which involves the removal of a short fragment on the C-terminus of SUMO inactive form and exposure two glycine residues. SENPs are important for maintaining the balance between sumoylated and desumoylated proteins required for normal cellular physiology. Six SENP isoforms (SENP1, SENP2, SENP3, SENP5, SENP6 and SENP7) have been identified in mammals. These SENPs can be divided into three subfamilies based on their sequence homology, substrate specificity and subcellular localization. Results of studies indicate the role of SUMO proteases in the development of human diseases including cancer, suggesting that these proteins may be attractive targets for new drugs.

P.4.f.007 - Subcortical brain volumes as endophenotypes of social anxiety disorder – preliminary findings from the Leiden Family Study on social anxiety disorder

Small ubiquitin-like modifier (SUMOylation) is a reversible post-translational modification, which plays important roles in numerous biological processes. SUMO could be covalently attached to target proteins in an isopeptide bond manner that occurs via a lysine ε-amino group on the target proteins and the glycine on SUMO C-terminus. This covalent binding could affect the subcellular localization and stability of target proteins. SUMO modification can be reversed by members of the Sentrin/SUMO-specific proteases (SENPs) family, which are highly evolutionarily conserved from yeast to human. SENP2, a member of the SENPs family, mainly plays a physiological function in the nucleus. SENP2 can promote maturity of the SUMO and deSUMOylate for single-SUMO modified or poly-SUMO modified proteins. SENP2 can affect the related biological processes through its peptidase activity or the amino terminal transcriptional repression domain. It plays important roles by inhibiting or activating some molecular functions. Therefore, the research achievements of SENP2 are reviewed in order to understand its related functions and the underlying molecular mechanisms and provide a clue for future research on SENP2.

Predictable Peptide Conjugation Ratios by Activation of Proteins with Succinimidyl Iodoacetate (SIA).

The small heterobifunctional linker succinimidyl iodoacetate (SIA) was examined for the preparation of peptide–protein bioconjugates with predicable conjugation ratios. For many conjugation protocols, the protein is either treated with a reductant to cleave disulfide bonds or is reacted with thiolation chemicals, such as Traut’s reagent. Both approaches are difficult to control, need individual optimization and often lead to unsatisfactory results. In another popular approach, a heterobifunctional linker with a N-hydroxysuccinimide (NHS) and a maleimide functionality is applied to the protein. After the activation of some lysine ε-amino groups with the NHS ester functionality, a cysteine-containing peptide is attached to the activated carrier protein via maleimide. Particularly, the maleimide reaction leads to some unwanted byproducts or even cleavage of the linker. Many protocols end up with conjugates with unpredictable and irreproducible conjugation ratios. In addition, the maleimide-thiol addition product should be assumed immunogenic in vivo. To avoid these and other disadvantages of the maleimide approach, we examined the known linker succinimidyl iodoacetate (SIA) in more detail and developed two protocols, which lead to peptide–protein conjugates with predefined average conjugation ratios. This holds potential to eliminate tedious and expensive optimization steps for the synthesis of a bioconjugate of optimal composition.

Hydroxyhomocitrulline Is a Collagen-Specific Carbamylation Mark that Affects Cross-link Formation

Carbamylation is a non-enzymatic post-translational modification that physiologically occurs during agingand is a risk factor for various diseases. The mostcommon product of carbamylation is homocitrulline (HCit), where a lysine (Lys) amino group hasreacted with urea-derived cyanate. HCit has recently beendetected in collagen; however, given that 15%-90% of total Lys in collagen is hydroxylated, it is unclear how hydroxylation affects collagen carbamylation. Here, we identified a collagen-specific carbamylation product, hydroxyhomocitrulline (HHCit), and showed that high levels of HHCit are correlated with age in rat tissue collagen and invivo carbamylation in mice, as well as with the decline of kidney function in the serum of dialysis patients. Proteomic analysis of the carbamylated collagens identified α2(I) Lys933, a major cross-linking site, as a preferential HHCit site. Furthermore, our results suggest that hydroxylysine carbamylation affects the mechanical properties of connective tissue by competitively inhibiting collagen cross-link formation.

Selective Reagent for Detection of N‐ε‐Monomethylation of a Peptide Lysine Residue through SNAr Reaction

Methylations of specific lysine residues of histone proteins are catalyzed by histone methyltransferases (HMTs) and play key roles in the epigenetic control of gene expression. Several methods to detect N‐ε‐methylation of the lysine residue have been established in order to evaluate the activity of HMTs, to develop inhibitors, and to identify substrates. However, they mostly employ specific antibodies or enzymes such as peptidases, and their reliability and reproducibility often depend on the quality of the protein reagents and the reaction conditions. Here, we describe a convenient method to detect N‐ε‐monomethylation of the lysine residue through a simple chemical reaction. We focused on nucleophilic aromatic substitution reaction (SNAr reaction) between an aromatic electrophile and a primary or monomethylated amino group. Screening of various electrophiles indicated that 4‐fluoro‐2‐nitroacetophenone (1g) has high selectivity for the N‐ε‐monomethylated amino group of lysine. Furthermore, the reaction products of 1g with lysine and N‐ε‐monomethylated lysine, 5g and 6g, respectively, show different absorption spectra, that is, the absorbance at 350 nm of 6g is 13 times larger than that of 5g. We show that these characteristic properties of 1g can be utilized for the selective detection of the methylation state of lysine residues in HMT substrate peptides, and for an assay of HMT activity.

Design of theranostic nanomedicine (II): synthesis and physicochemical properties of a biocompatible polyphosphazene–docetaxel conjugate

To prepare an efficient theranostic polyphosphazene–docetaxel (DTX) conjugate, a new drug delivery system was designed by grafting a multifunctional lysine ethylester (LysOEt) as a spacer group along with methoxy poly(ethylene glycol) (MPEG) to the polyphosphazene backbone ([NP]n), and then DTX was conjugated to the carrier polymer using acid-cleavable cis-aconitic acid (AA) as a linker. The resultant polyphosphazene–DTX conjugate, formulated as [NP(MPEG550)3(Lys-OEt)(AA)(DTX)]n and named “Polytaxel”, exhibited high water solubility and stability by forming stable polymeric micelles as shown in its transmission electron microscopy image and dynamic light scattering measurements. Another important aspect of Polytaxel is that it can easily be labeled with various imaging agents using the lysine amino group, enabling studies on various aspects, such as its organ distribution, tumor-targeting properties, pharmacokinetics, toxicity, and excretion. The pharmacokinetics of Polytaxel was remarkably improved, with prolonged elimination half-life and enhanced area under the curve. Ex vivo imaging study of cyanine dye-labeled Polytaxel showed that intravenously injected Polytaxel is long circulating in the blood stream and selectively accumulates in tumor tissues. Polytaxel distributed in other organs was cleared from all major organs at ~6 weeks after injection. The in vitro study of DTX release from the carrier polymer showed that >95% of conjugated DTX was released at pH 5.4 over a period of 7 days. Xenograft trials of Polytaxel using nude mice against the human gastric tumor cell line MKN-28 showed complete tumor regression, with low systemic toxicity. Polytaxel is currently in preclinical study.

Destructive effect of non-enzymatic glycation on catalase and remediation via curcumin

Non-enzymatic glycation of proteins is a post-translational modification that is produced by a covalent binding between reducing sugars and amino groups of lysine and arginine residues. In this paper the effect of pathological conditions, derived from hyperglycemia on bovine liver catalase (BLC) as a model protein was considered by measuring enzyme activity, reactive oxygen species (ROS) generation, and changes in catalase conformational properties. We observed that in the presence of glucose, the catalase activity gradually decreased. ROS generation was also involved in the glycation process. Thus, decreased BLC activity was partly considered as a result of ROS generation through glycation. However, in the presence of curcumin the amount of ROS was reduced resulting in increased activity of the glycated catalase. The effect of high glucose level and the potential inhibitory effect of curcumin on aggregation and structural changes of catalase were also investigated. Molecular dynamic simulations also showed that interaction of catalase with curcumin resulted in changes in accessible surface area (ASA) and pKa, two effective parameters of glycation, in potential glycation lysine residues. Thus, the decrease in ASA and increase in pKa of important lysine residues were considered as predominant factors in decreased glycation of BLC by curcumin.

The ND EarlyQPM program: developing the next generation of healthier maize (Zea mays L.) products

There is a need to develop the next generation of healthier crop products for an improved human and animal nutrition. Maize (Zea mays L.) cultivars with improved amino acid profiles are essential to diets focused on this crop. Breeders have added crop value with the development of quality protein maize (QPM). However, QPM cultivars have never been an option to farmer and ranchers in short-season environments. The objectives of this research were: (1) to adapt QPM genotypes to the northern U.S. through the North Dakota (ND) EarlyQPM Program, and (2) to develop new early generation SS and non-SS short-season QPM lines and populations for breeding purposes. Fifty-three inbred lines, including 47 QPM donor lines, five experimental North Dakota State University (NDSU) lines, and one ex-PVP line from industry, were selected to produce 94 early-QPM backcross populations. Considering earliness, protein content, and amino acid levels, 218 early generation lines were selected for producing testcrosses with industry testers. Experiments evaluating testcrosses were arranged in 10 × 10 and 12 × 12 partially balanced lattice designs across three ND locations in 2013 and 2014. A total of 48 lines were selected for further development, 17 representing the Stiff Stalk (SS) heterotic group and 31 representing the non-SS group. Selected lines showed, in hybrid combinations, not only above average grain yield, rate of dry down, and total protein content but also high levels of lysine, tryptophan, and methionine essential amino acids for feedstock nutrition. The results of this research show, for the first time, the successful adaptation of QPM genotypes to short-season environments. As a result, new ND EarlyQPM germplasms and lines have been developed for potential release.

Acetylation of lysine ϵ-amino groups regulates aminoacyl-tRNA synthetase activity in Escherichia coli

Previous proteomic analyses have shown that aminoacyl-tRNA synthetases in many organisms can be modified by acetylation of Lys. In this present study, leucyl-tRNA synthetase and arginyl-tRNA synthetase from Escherichia coli (EcLeuRS and EcArgRS) were overexpressed and purified and found to be acetylated on Lys residues by MS. Gln scanning mutagenesis revealed that Lys619, Lys624, and Lys809 in EcLeuRS and Lys126 and Lys408 in EcArgRS might play important roles in enzyme activity. Furthermore, we utilized a novel protein expression system to obtain enzymes harboring acetylated Lys at specific sites and investigated their catalytic activity. Acetylation of these Lys residues could affect their aminoacylation activity by influencing amino acid activation and/or the affinity for tRNA. In vitro assays showed that acetyl-phosphate nonenzymatically acetylates EcLeuRS and EcArgRS and suggested that the sirtuin class deacetylase CobB might regulate acetylation of these two enzymes. These findings imply a potential regulatory role for Lys acetylation in controlling the activity of aminoacyl-tRNA synthetases and thus protein synthesis.

Unique Pattern of Protein-Bound Maillard Reaction Products in Manuka (Leptospermum scoparium) Honey.

As a unique feature, honey from the New Zealand manuka tree (Leptospermum scoparium) contains substantial amounts of dihydroxyacetone (DHA) and methylglyoxal (MGO). Although MGO is a reactive intermediate in the Maillard reaction, very little is known about reactions of MGO with honey proteins. We hypothesized that the abundance of MGO should result in a particular pattern of protein-bound Maillard reaction products (MRPs) in manuka honey. A protein-rich high-molecular-weight fraction was isolated from 12 manuka and 8 non-manuka honeys and hydrolyzed enzymatically. By HPLC-MS/MS, 8 MRPs, namely, N-ε-fructosyllysine, N-ε-maltulosyllysine, carboxymethyllysine, carboxyethyllysine (CEL), pyrraline, formyline, maltosine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1), were quantitated. Compared to non-manuka honeys, the manuka honeys were characterized by high concentrations of CEL and MG-H1, whereas the formation of N-ε-fructosyllysine was suppressed, indicating concurrence reactions of glucose and MGO at the ε-amino group of protein-bound lysine. Up to 31% of the lysine and 8% of the arginine residues, respectively, in the manuka honey protein can be modified to CEL and MG-H1, respectively. CEL and MG-H1 concentrations correlated strongly with the MGO concentration of the honeys. Manuka honey possesses a special pattern of protein-bound MRPs, which might be used to prove the reliability of labeled MGO levels in honeys and possibly enable the detection of fraudulent MGO or DHA addition to honey.

Mass Spectrometry Based Proteomics Study of Cisplatin-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells.

Platinum-based antitumor drugs such as 1,1,2,2-cis-diamminedichloroplatinum(II) (cisplatin), carboplatin, and oxaliplatin are currently used to treat nearly 50% of all cancer cases, and novel platinum based agents are under development. The antitumor effects of cisplatin and other platinum compounds are attributed to their ability to induce interstrand DNA-DNA cross-links, which are thought to inhibit tumor cell growth by blocking DNA replication and/or preventing transcription. However, platinum agents also induce significant numbers of unusually bulky and helix-distorting DNA-protein cross-links (DPCs), which are poorly characterized because of their unusual complexity. We and others have previously shown that DPCs block DNA replication and transcription and causes toxicity in human cells, potentially contributing to the biological effects of platinum agents. In the present work, we have undertaken a system-wide investigation of cisplatin-mediated DNA-protein cross-linking in human fibrosarcoma (HT1080) cells using mass spectrometry-based proteomics. DPCs were isolated from cisplatin-treated cells using a modified phenol/chloroform DNA extraction in the presence of protease inhibitors. Proteins were released from DNA strands and identified by mass spectrometry-based proteomics and immunological detection. Over 250 nuclear proteins captured on chromosomal DNA following treatment with cisplatin were identified, including high mobility group (HMG) proteins, histone proteins, and elongation factors. To reveal the exact molecular structures of cisplatin-mediated DPCs, isotope dilution HPLC-ESI+-MS/MS was employed to detect 1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) conjugates between the N7 guanine of DNA and the ε-amino group of lysine. Our results demonstrate that therapeutic levels of cisplatin induce a wide range of DPC lesions, which likely contribute to both target and off target effects of this clinically important drug.

Intranasal delivery of N-terminal modified leptin-pluronic conjugate for treatment of obesity

Leptin is an adipocyte-secreted hormone that is delivered via a specific transport system across the blood-brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose-to-brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity.

174 Effects of dietary lysine level and amino acid ratios on nursery pig performance

174 Effects of dietary lysine level and amino acid ratios on nursery pig performance

Palladium‐Mediated Arylation of Lysine in Unprotected Peptides

AbstractA mild method for the arylation of lysine in an unprotected peptide is presented. In the presence of a preformed biarylphosphine‐supported palladium(II)–aryl complex and a weak base, lysine amino groups underwent C−N bond formation at room temperature. The process generally exhibited high selectivity for lysine over other amino acids containing nucleophilic side chains and was applicable to the conjugation of a variety of organic compounds, including complex drug molecules, with an array of peptides. Finally, this method was also successfully applied to the formation of cyclic peptides by macrocyclization.

Palladium-Mediated Arylation of Lysine in Unprotected Peptides.

A mild method for the arylation of lysine in an unprotected peptide is presented. In the presence of a preformed biarylphosphine-supported palladium(II)-aryl complex and a weak base, lysine amino groups underwent C-N bond formation at room temperature. The process generally exhibited high selectivity for lysine over other amino acids containing nucleophilic side chains and was applicable to the conjugation of a variety of organic compounds, including complex drug molecules, with an array of peptides. Finally, this method was also successfully applied to the formation of cyclic peptides by macrocyclization.

Immunoglobulin-G Glycation by Fructose Leads to Structural Perturbations and Drop Off in Free Lysine and Arginine Residues.

Non-enzymatic glycation is the addition of free carbonyl group of reducing sugar to the free amino groups of proteins and leads to the formation of early glycation products and further into advanced glycation end products (AGEs). Fructose reacts rapidly with the free amino groups of proteins to form AGEs. AGEs are believed to be involved in the pathogenesis of several diseases, particularly in diabetic complications. In this study, IgG was glycated with fructose monosaccharide at 10 mM concentration for varying time interval. The reaction mixture was kept at 37ºC. The early glycation of IgG was done by nitroblue tetrazolium assay (NBT), and the generation of AGEs was done by the extent of side chain modifications (lysine and arginine), Nε-carboxymethyl lysine, pentosidine and carbonyl content. The decrease in free lysine and arginine residues suggests that protein 'IgG' has undergone modification specifically on epsilon amino groups of lysine and arginine. Additionally, their fluorescence and absorbance characteristics were also systematically studied. The results suggest that the maximum Amadori product (ketoamine content) was formed on sixth day of the incubation. The conformational structural perturbation was observed within the glycated IgG protein as studied by using various physicochemical techniques. This study reports structural perturbation, formation of various intermediates and AGEs.

The Current State of NAD+ -Dependent Histone Deacetylases (Sirtuins) as Novel Therapeutic Targets.

Sirtuins are NAD+ -dependent protein deacylases that cleave off acetyl, as well as other acyl groups, from the ε-amino group of lysines in histones and other substrate proteins. Seven sirtuin isotypes (Sirt1-7) have been identified in mammalian cells. As sirtuins are involved in the regulation of various physiological processes such as cell survival, cell cycle progression, apoptosis, DNA repair, cell metabolism, and caloric restriction, a dysregulation of their enzymatic activity has been associated with the pathogenesis of neoplastic, metabolic, infectious, and neurodegenerative diseases. Thus, sirtuins are promising targets for pharmaceutical intervention. Growing interest in a modulation of sirtuin activity has prompted the discovery of several small molecules, able to inhibit or activate certain sirtuin isotypes. Herein, we give an update to our previous review on the topic in this journal (Schemies, 2010), focusing on recent developments in sirtuin biology, sirtuin modulators, and their potential as novel therapeutic agents.

Peptides Labeled with Pyridinium Salts for Sensitive Detection and Sequencing by Electrospray Tandem Mass Spectrometry.

Mass spectrometric analysis of trace amounts of peptides may be problematic due to the insufficient ionization efficiency resulting in limited sensitivity. One of the possible ways to overcome this problem is the application of ionization enhancers. Herein we developed new ionization markers based on 2,4,6-triphenylpyridinium and 2,4,6-trimethylpyridinium salts. Using of inexpensive and commercially available pyrylium salt allows selective derivatization of primary amino groups, especially those sterically unhindered, such as ε-amino group of lysine. The 2,4,6-triphenylpyridinium modified peptides generate in MS/MS experiments an abundant protonated 2,4,6-triphenylpyridinium ion. This fragment is a promising reporter ion for the multiple reactions monitoring (MRM) analysis. In addition, the fixed positive charge of the pyridinium group enhances the ionization efficiency. Other advantages of the proposed ionization enhancers are the simplicity of derivatization of peptides and the possibility of convenient incorporation of isotopic labels into derivatized peptides.

Insights into the Biosynthesis of Duramycin.

Duramycin is a natural product produced by certain bacteria that binds to phosphatidylethanolamine (PE). Because PE is involved in many cellular processes, duramycin is an antibiotic that kills bacteria, but it has also been used as a molecular probe to detect PE and monitor its localization in mammalian cells and even whole organisms, and it was recently shown to display broad-spectrum inhibition of viral entry into host cells. In addition, the molecule has been evaluated as treatment for cystic fibrosis. We report here the genes that are involved in duramycin biosynthesis, and we produced duramycin by expressing those genes in Escherichia coli We show that duramycin analogs can also be produced. The ability to access duramycin and analogs by production in E. coli opens opportunities to improve duramycin as an antibiotic, PE probe, antiviral, or cystic fibrosis therapeutic.

Elastin structure and its involvement in skin photoageing

Skin aging is a complex process that may be caused by factors that are intrinsic and extrinsic to the body. Ultraviolet (UV) radiation represents one of the main sources of skin damage over the years and characterizes a process known as photoaging. Among the changes that affect cutaneous tissue with age, the loss of elastic properties caused by changes in elastin production, increased degradation and/or processing produces a substantial impact on tissue esthetics and health. The occurrence of solar elastosis is one of the main markers of cutaneous photoaging and is characterized by disorganized and non-functional deposition of elastic fibers. The occurrence of UV radiation-induced alternative splicing of the elastin gene, which leads to inadequate synthesis of the proteins required for the correct assembly of elastic fibers, is a potential explanation for this phenomenon. Innovative studies have been fundamental for the elucidation of rarely explored photoaging mechanisms and have enabled the identification of effective therapeutic alternatives such as cosmetic products. This review addresses cutaneous photoaging and the changes that affect elastin in this process.