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Background: Loss of function variants in the ornithine aminotransferase ( OAT) gene cause accumulation of ornithine levels, leading to gyrate atrophy. The benefit of ornithine-lowering therapies has been documented in a mouse model and young patients, however, the effect in adults with advanced disease has not been well described., Materials and Methods: Case report of an adult patient with advanced gyrate atrophy, who underwent treatment with pyridoxine and an arginine-restricted diet for four years., Results: A 51-year-old female with advanced chorioretinal degeneration presented with hyperornithinemia (961 vs. normal 18-135 µmol/L) and compound heterozygous pathogenic variants in OAT (p.Tyr299* and p.Ala270Pro). Treatment with pyridoxine and arginine-diet restriction yielded a maximal reduction in ornithine levels by 71% (275 µmol/L). Optical coherence tomography (OCT) showed a reduction in ellipsoid zone (EZ) thickness that correlated with lower ornithine levels and reversed with higher ornithine levels. While her best-corrected visual acuity remained unchanged, the progressive decline in her visual fields appeared to stabilize during a one-year period when ornithine levels were below 500 µmol/L., Conclusions: In this report, we demonstrate that chorioretinal degeneration appears to stabilize in an adult patient with gyrate atrophy in association with a partial reduction in ornithine levels. We also observed a correlation with reduced EZ thickness on OCT and propose this may be a novel biomarker for ornithine reduction therapies. Our case study characterizes the potential retinal structure-function benefits of ornithine-lowering treatments even in cases of advanced chorioretinal degeneration. Thus, we recommend a low threshold for treating all patients with gyrate atrophy.

Protein methylation is one of the most prominent posttranslation modifications that essentially regulates several biological processes in eukaryotes. Therefore, identification of the arginine methylation site is crucial in deciphering its characteristics and functions in cell biology, disease mechanisms, and guided drug development. The computation methods address the long-term bottleneck together with the cost, time, and labor required in experimental methods for large-scale identification of protein arginine methylation sites. In this study, we proposed a robust machine learning-based computational tool known as iIRMethyl, employing the primary sequence and physicochemical properties of protein along with a two-step feature selection method for optimal selection of feature descriptors. Moreover, the performance of iIRMethyl was comprehensively evaluated via k-fold cross-validation on a benchmark dataset and independent test dataset. iIRMethyl demonstrated a remarkably greater performance than the state-of-the-art method and achieved an average area under the curve value of 0.99 for both k-fold cross-validation and an independent test set in the identification of protein arginine methylation sites. Furthermore, the outcomes reveal that iIRMethyl is a robust and accurate computational tool for large-scale identification of arginine methylation sites and would facilitate the understanding of their functional mechanisms and accelerating their application in drug development and clinical therapy. Additionally, the prediction mechanism of the proposed model iIRMethyl is interpreted using the SHapley Additive exPlanation algorithm., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Methylated free amino acids are an important class of targets for host-guest chemistry that have recognition properties distinct from those of methylated peptides and proteins. We present comparative binding studies for three different host classes that are each studied with multiple methylated arginines and lysines to determine fundamental structure-function relationships. The hosts studied are all anionic and include three calixarenes, two acyclic cucurbiturils, and two other cleft-like hosts, a clip and a tweezer. We determined the binding association constants for a panel of methylated amino acids using indicator displacement assays. The acyclic cucurbiturils display stronger binding to the methylated amino acids, and some unique patterns of selectivity. The two other cleft-like hosts follow two different trends, shallow host (clip) following similar trends to the calixarenes, and the other more closed host (tweezer) binding certain less-methylated amino acids stronger than their methylated counterparts. Molecular modelling sheds some light on the different preferences of the various hosts. The results identify hosts with new selectivities and with affinities in a range that could be useful for biomedical applications. The overall selectivity patterns are explained by a common framework that considers the geometry, depth of binding pockets, and functional group participation across all host classes., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

The integral membrane, Kunitz-type, serine protease inhibitors, HAI-1 and HAI-2, closely resemble one another structurally and with regard to their specificity and potency against proteases. Structural complementarity between the Kunitz domains and serine protease domains renders the membrane-associated serine proteases, matriptase and prostasin, the primary target proteases of the HAIs. The shared biochemical enzyme-inhibitor relationships are, however, at odds with their behavior at the cellular level, where HAI-1 appears to be the default inhibitor of these proteases and HAI-2 a cell-type-selective inhibitor, even though they are widely co-expressed. The limited motility of these proteins caused by their membrane anchorages may require their co-localization within a certain distance to allow the establishment of a cellular level functional relationship between the proteases and the inhibitors. The differences in their subcellular localization with HAI-1 both inside the cell and on the cell surface, compared to HAI-2 predominately in intracellular granules has, therefore, been implicated in the differential manner of their control of matriptase and prostasin proteolysis. The targeting signals present in the intracellular domains of the HAIs are systematically investigated herein. Studies involving domain swap and point mutation, in combination with immunocytochemistry and cell surface biotinylation/avidin depletion, reveal that the different subcellular localization between the HAIs can largely be attributed to differences in the intracellular Arg/Lys-rich and EHLVY motifs. These intrinsic differences in the targeting signal render the HAIs as two independent rather than redundant proteolysis regulators., (© 2021. Japan Human Cell Society.)

Lysosomes degrade excess or damaged cellular components and recycle their building blocks through membrane transporters. They also act as nutrient-sensing signaling hubs to coordinate cell responses. The membrane protein PQ-loop repeat-containing protein 2 (PQLC2; "picklock two") is implicated in both functions, as it exports cationic amino acids from lysosomes and serves as a receptor and amino acid sensor to recruit the C9orf72/SMCR8/WDR41 complex to lysosomes upon nutrient starvation. Its transport activity is essential for drug treatment of the rare disease cystinosis. Here, we quantitatively studied PQLC2 transport activity using electrophysiological and biochemical methods. Charge/substrate ratio, intracellular pH, and reversal potential measurements showed that it operates in a uniporter mode. Thus, PQLC2 is uncoupled from the steep lysosomal proton gradient, unlike many lysosomal transporters, enabling bidirectional cationic amino acid transport across the organelle membrane. Surprisingly, the specific presence of arginine, but not other substrates (lysine, histidine), in the discharge (" trans ") compartment impaired PQLC2 transport. Kinetic modeling of the uniport cycle recapitulated the paradoxical substrate-yet-inhibitor behavior of arginine, assuming that bound arginine facilitates closing of the transporter's cytosolic gate. Arginine binding may thus tune PQLC2 gating to control its conformation, suggesting a potential mechanism for nutrient signaling by PQLC2 to its interaction partners., Competing Interests: The authors declare no competing interest. See online for related content such as Commentaries., (Copyright © 2021 the Author(s). Published by PNAS.)

Biological aging research is expected to reveal modifiable molecular mechanisms that can be harnessed to slow or possibly reverse unhealthy trajectories. However, there is first an urgent need to define consensus molecular markers of healthy and unhealthy aging. Established aging hallmarks are all linked to metabolism, and a 'rewired' metabolic circuitry has been shown to accelerate or delay biological aging. To identify metabolic signatures distinguishing healthy from unhealthy aging trajectories, we performed nontargeted metabolomics on skeletal muscles from 2-month-old and 21-month-old mice, and after dietary and lifestyle interventions known to impact biological aging. We hypothesized that common metabolic signatures would highlight specific pathways and processes promoting healthy aging, while revealing the molecular underpinnings of unhealthy aging. Here, we report 50 metabolites that commonly distinguished aging trajectories in all cohorts, including 18 commonly reduced under unhealthy aging and 32 increased. We stratified these metabolites according to known relationships with various aging hallmarks and found the greatest associations with oxidative stress and nutrient sensing. Collectively, our data suggest interventions aimed at maintaining skeletal muscle arginine and lysine may be useful therapeutic strategies to minimize biological aging and maintain skeletal muscle health, function, and regenerative capacity in old age.

Positively charged amino acid side-chains play important roles in anion binding and permeation through the CFTR chloride channel. One pore-lining lysine residue in particular (K95) has been shown to be indispensable for anion binding, conductance, and selectivity. Here, we use functional investigation of CFTR to show that a nearby arginine (R134) plays a functionally analogous role. Removal of this positive charge (in the R134Q mutant) drastically reduces single-channel conductance, weakens binding of both permeant and blocking anions, and abolishes the normal anion conductance selectivity pattern. Each of these functional effects was reversed by a second-site mutation (S1141K) that introduces an ectopic positive charge to a nearby pore-lining residue. Substituted cysteine accessibility experiments confirm that R134-but not nearby residues in the same transmembrane helix-is accessible within the pore lumen. These results suggest that K95 and R134, which are very close together within the inner vestibule of the pore, play analogous, important roles, and that both are required for the normal anion binding and anion conductance properties of the pore. Nevertheless, that fact that both positive charges can be "transplanted" to other sites in the inner vestibule with little effect on channel permeation properties indicates that it is the overall number of charges-rather than their exact locations-that controls pore function.

The accumulation of advanced glycation end-products is a fundamental process that is central to age-related decline in musculoskeletal tissues and locomotor system function and other collagen-rich tissues. However, although computational studies of advanced glycation end-product cross-links could be immensely valuable, this area remains largely unexplored given the limited availability of structural parameters for the derivation of force fields for Molecular Dynamics simulations. In this article, we present the bonded force constants, atomic partial charges and geometry of the arginine-lysine cross-links DOGDIC, GODIC, and MODIC. We have performed in vacuo Molecular Dynamics simulations to validate their implementation against quantum mechanical frequency calculations. A DOGDIC advanced glycation end-product cross-link was then inserted into a model collagen fibril to explore structural changes of collagen and dynamics in interstitial water. Unlike our previous studies of glucosepane, our findings suggest that intra-collagen DOGDIC cross-links furthers intra-collagen peptide hydrogen-bonding and does not promote the diffusion of water through the collagen triple helices., (© 2020 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

AKT/PKB is downregulated by the ubiquitin-proteasome system (UPS), which plays a key role in cell survival and tumor progression in various types of cancer. The objective of this study was to determine the relationship between the sequential ubiquitination of lysine residues K284 to K214 in AKT and R-HSPA5 (the arginylated form of HSPA5), which contribute to the autophagic/lysosomal degradation of AKT when impaired proteasomal activity induces cellular stress. Results show that proteasome inhibitors (PIs) increased ATE1 (arginyltransferase 1)-mediated R-HSPA5 levels in a reactive oxygen species (ROS)-dependent manner. Further, binding of fully ubiquitinated AKT with R-HSPA5 induced AKT degradation via the autophagy-lysosome pathway. Specifically, the K48 (Lys48)-linked ubiquitinated form of AKT was selectively degraded in the lysosome with R-HSPA5. The deubiquitinase, USP7 (ubiquitin specific peptidase 7), prevented AKT degradation by inhibiting AKT ubiquitination via interaction with AKT. MUL1 (mitochondrial ubiquitin ligase activator of NFKB 1) also played a vital role in the lysosomal degradation of AKT by sequentially ubiquitinating AKT residues K284 to K214 for R-HSPA5-mediated autophagy. Consistent with this finding, despite HSPA5 arginylation, AKT was not degraded in mul1 KO cells. These results suggest that MUL1-mediated sequential ubiquitination of K284 to K214 may serve as a novel mechanism by which AKT is designated for lysosomal degradation. Moreover, binding of R-HSPA5 with fully ubiquitinated AKT is required for the autophagic/lysosomal degradation of AKT. Thus, modulating the MUL1-mediated non-proteasomal proteolysis mechanisms, such as sequential ubiquitination, may prove to be a novel therapeutic approach for cancer treatment. Abbreviations : AKT1: thymoma viral proto-oncogene 1; ATE1: arginyltransferase 1; ATG5: autophagy related 5; CASP3: caspase 3; EGFP: enhanced green fluorescent protein; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3B; glycogen synthase kinase 3 beta; HA: hemagglutinin; HSPA5/GRP78/BIP: heat shock protein 5; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MUL1: mitochondrial ubiquitin ligase activator of NFKB1; NAC: N-acetylcysteine; NEK2: NIMA (never in mitosis gene a)-related expressed kinase 2; NH 4 Cl: ammonium chloride; PARP1: poly(ADP-ribose) polymerase family, member 1; PI: proteasome inhibitor; R-HSPA5: arginylated HSPA5; ROS: reactive oxygen species; SQSTM1: sequestome 1; Ub: ubiquitin; USP7: ubiquitin specific peptidase 7.

The stm1 + (SPAC17C9.10) gene of Schizosaccharomyces pombe is closely related to genes encoding vacuolar PQ-loop proteins, Ypq1, Ypq2, and Ypq3, of Saccharomyces cerevisiae. When stm1 + fused with GFP was expressed in fission or budding yeast, Stm1-GFP localized at the vacuolar membrane. Isolated vacuolar membrane vesicles from S. cerevisiae cells overexpressing stm1 + exhibited stm1 + -dependent arginine and lysine uptake activity. Exchange activity of arginine and histidine/arginine, as observed for Ypq2 of S. cerevisiae, was also detected in the vesicles expressing stm1 + . The expression levels of stm1 + in S. pombe cells significantly affected the vacuolar contents of lysine, histidine, and arginine. These results suggest that Stm1 is a vacuolar PQ-loop protein involved in the transport of basic amino acids across the vacuolar membrane., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We postulated that the use of appropriate levels and proportions of arginine (Arg) and methionine (Met) in compound feed with high lysine content (Lys) would make it possible to fully exploit the growth potential of modern fattening turkey crossbreds, without compromising their immune system. The aim of this study was to determine the effect of different ratios of Arg and Met in diets with high Lys content on the performance and immune status of turkeys. The turkeys were assigned to 6 groups with 8 replicates per group and 18 birds per replicate. Six feeding programs, with 3 dietary Arg levels (90, 100, and 110%) and 2 dietary Met levels (30 and 45%) relative to dietary Lys content, were compared. During each of 4 feeding phases (weeks 0-4, 5-8, 9-12, and 13-16), birds were fed ad libitum isocaloric diets containing high level of Lys, approximately 1.83, 1.67, 1.49, and 1.20%, respectively. The dietary treatments had no effect on daily feed intake or body weight at any stage of the study. The protein content of the breast meat was higher in the treatments with the highest Arg level (110%) compared with the lowest Arg level (90%). Similarly, protein content was higher in the treatments with the higher Met level compared with the lower Met level. Higher plasma levels of tumor necrosis factor, interleukin 6 (IL-6), and immunoglobulin Y were found in turkeys fed diets with the lowest Arg content. An increase in Met content resulted in a decrease in plasma content of IL-6. In growing turkeys fed diets high in Lys, an Arg level of 90% relative to Lys can be used without negatively affecting production results and immune system. Regardless of dietary Arg levels, an increase in Met content does not stimulate the immune defense system and shows no effect on growth performance of turkeys in current trial., (Copyright © 2020. Published by Elsevier Inc.)

The present study investigated the effect of optimizing the total dietary arginine (Arg)-to-lysine (Lys) ratios on the metabolism of lactating sows and piglet performance by supplementation with l- Arg during lactation. A total of 200 multiparous sows (three to six parities, Yorkshire × Landrace) were selected and randomly and equally assigned to five groups in lactation, and finally, 36, 34, 35, 36, and 33 dams completed the study in the dietary treatments, respectively, where the diets consisted of five step-up Arg-to-Lys ratios (0.9, 1.0, 1.1, 1.2, and 1.3) by the addition of 0%, 0.10%, 0.20%, 0.30%, and 0.40% Arg. The diets contained 3.37 to 3.38 Mcal of digestible energy/kg energy, 17.73% to 17.75% crude protein, and 0.98% to 1.01% Lys and were fed ad libitum during lactation. The performance of sows and suckling piglets was measured, and plasma and milk samples were collected for analysis. The feed intake of sows as well as litter weight gain during lactation increased linearly (P ≤ 0.05), while maternal backfat and milk composition were not affected (P > 0.05) as the dietary Arg-to-Lys ratios increased. Analyzed plasma biochemical indices, including concentrations of free Arg, Orn, and Glu, and prolactin, insulin, and follicle-stimulating hormone, responded linearly (P ≤ 0.05) to increases in dietary Arg-to-Lys ratios. The dietary Arg-to-Lys ratios of 1.01 and 1.02 were optimal for maternal feed intake and litter weight gain, based on broken-line models. Collectively, the results of this study indicate that increasing total dietary Arg-to-Lys ratios in lactation was beneficial for the performance of lactating sows and suckling piglets, and dietary Arg-to-Lys ratios of 1.01 and 1.02 were optimal, from regression analyses, for the practical feeding of lactating sows., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Patients with lysine-related inborn errors of metabolism (pyridoxine-dependent epilepsy [PDE] and glutaric aciduria type 1 [GA1]), follow a lysine-restricted diet with arginine-fortified lysine-free amino acid formula and additional oral arginine supplementation as a newer therapy for PDE. The rationale of arginine supplementation is based on arginine's ability to compete with lysine transport across cell membranes via shared transporter systems. Adequate doses of arginine required to competitively inhibit enteral lysine uptake has not been studied in humans This proof-of-concept study investigates the effect of incremental enteral arginine doses on whole-body lysine oxidation using an in vivo stable isotope tracer, L-[1- 13 C] lysine, in healthy humans. Five healthy men completed six study days each consuming one dose of l-arginine HCl per study day; range = 50-600 mg/kg/d. Lysine intake was at DRI (30 mg/kg/d). Breath samples were analysed for L-[1- 13 C] lysine oxidation to 13 CO 2 using an isotope ratio mass spectrometer. Plasma amino acid concentrations were analysed using an amino acid analyser. Increasing doses of l-arginine HCl caused a linear decrease in whole-body lysine oxidation. Plasma arginine concentration increased, and plasma lysine concentration decreased below normal range with high arginine intakes. We provide the first empirical evidence of arginine-lysine antagonism in response to increasing oral arginine doses. Results suggest 300-600 mg/kg/d of l-arginine HCl and lysine intake restricted to DRI is needed to reduce enteral lysine uptake and systemic lysine oxidation. This could potentially lead to a recommended dose for arginine in lysine-related inborn errors of metabolism., (© 2020 SSIEM.)

Hsp16.3, a molecular chaperone, plays a vital role in the growth and survival of Mycobacterium tuberculosis inside the host. We previously reported that deletion of three amino acid residues ( 142 STN 144 ) from C-terminal extension (CTE) of Hsp16.3 triggers its structural perturbation and increases its chaperone activity, which reaches its apex upon the deletion of its entire CTE ( 141 RSTN 144 ). Thus, we hypothesized that Arg141 (R141) and Ser142 (S142) in the CTE of Hsp16.3 possibly hold the key in maintaining its native-like structure and chaperone activity. To test this hypothesis, we generated two deletion mutants in which R141 and S142 were deleted individually (Hsp16.3ΔR141 and Hsp16.3ΔS142) and three substitution mutants in which R141 was replaced by lysine (Hsp16.3R141K), alanine (Hsp16.3R141A), and glutamic acid (Hsp16.3R141E), respectively. Hsp16.3ΔS142 or Hsp16.3R141K mutant has native-like structure and chaperone activity. Deletion of R141 from the CTE (Hsp16.3ΔR141) perturbs the secondary and tertiary structure, lowers the subunit exchange dynamics and decreases the chaperone activity of Hsp16.3. But, the substitution of R141 with alanine (Hsp16.3R141A) or glutamic acid (Hsp16.3R141E) perturbs its secondary and tertiary structure. Surprisingly, such charge tampering of R141 enhances the subunit exchange dynamics and chaperone activity of Hsp16.3. Interestingly, neither the deletion of R141/S142 nor the substitution of R141 with lysine, alanine and glutamic acid affects the oligomeric mass/size of Hsp16.3. Overall, our study suggests that R141 (especially the positive charge on R141) plays a crucial role in maintaining the native-like structure as well as in regulating subunit exchange dynamics and chaperone activity of Hsp16.3., (© 2019 Wiley Periodicals, Inc.)

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.

Two experiments were performed to investigate the effect of different ratios of arginine (Arg) to lysine (Lys) in diets with low (30% Lys; Experiment 1) and high (45% Lys; Experiment 2) methionine (Met) levels on selected metabolic parameters, oxidative and epigenetic DNA damage, and the mechanisms underlying intestinal barrier integrity in turkeys challenged with Clostridium perfringens. In each experiment, 108 one-day-old Hybrid Converter female turkeys were placed in 6 pens (18 birds per pen) and reared for 42 days. At 34, 36 and 37 days of age, half of the birds were subjected to C. perfringens challenge. A 3 × 2 factorial design with three levels of Arg relative to Lys (90, 100 and 110%; Arg 90 , Arg 100 and Arg 110 , respectively) and C. perfringens infection (-, +) was employed. Challenging birds with C. perfringens increased lipid oxidation and the oxidation and methylation of DNA of intestinal mucosa, and down-regulated the activities of DNA-repairing enzymes. Neither the dietary treatment nor the challenge affected the markers of liver function or metabolism. Arg 110 diets with the high Met level induced DNA oxidation and methylation whereas these processes were downregulated in birds fed Arg 90 diets. The results indicate that Arg 90 diets with high Met levels have a beneficial influence on the indicators of intestinal barrier integrity in turkeys with necrotic enteritis (NE). Despite the analyzed amino acid ratios interacted with the systems responsible for the maintenance of gut integrity in the host organism, this dietary intervention probably enabled birds to cope with NE.

Age is an important parameter for a variety of ecological applications, including population viability analyses, contaminants monitoring and targeting of individuals for conservation. While many organisms can be aged by annual rings, dentition and other techniques (i.e., fish otoliths, clam growth rings, mammal tooth wear), there are no minimally invasive biomarkers for accurately aging birds in the wild. For the past century, banding has been the only way to identify a bird of known age, which requires continuous effort on a large scale with possibly low return rates. Recent studies have identified pentosidine as a potential biomarker of chronological aging in several bird species. To test this idea in four species of long-lived seabirds, we collected skin biopsies from the foot webs of previously banded, known-age seabirds: black-legged kittiwakes (Rissa tridactyla; 0-19 y old), Atlantic puffins (Fratercula arctica; 5-26 y old), razorbills (Alca torda; 0-15 d old) and thick-billed murres (Uria lomvia; 0-35 y old). Foot web samples were specifically chosen because this was the least invasive site for substantial skin biopsy. Samples were analysed with high performance liquid chromatography to quantify pentosidine levels. Collagen levels were estimated through hydroxyproline assays to normalize pentosidine content across individuals. Kittiwakes displayed a weak correlation (r 2  = 0.20) between age and pentosidine/collagen. Puffins (adults only, r 2  = 0.02), razorbills (chicks only, r 2  = 0.08), and murres (adults, r 2  = 0.04) did not show any associations with age. We concluded that pentosidine content in the foot web does not appear to be a reliable method for aging seabirds in the wild. An absence of change in pentosidine in the foot web with age is further evidence that long-lived seabirds may maintain physiological performance into old age., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Low-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine-aspartic acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine's guanidinium ion. MCDs can synergize with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behavior is tunable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. Contrastingly, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation and link the dynamic material properties of speckles with function in mRNA processing., Competing Interests: Declaration of Interests R.V.P. is a member of the Scientific Advisory Board of Dewpoint Therapeutics Inc. There are no competing financial or ethical conflicts to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The research hypothesis postulated that the optimal dietary inclusion levels and ratios of lysine (Lys), arginine (Arg), and methionine (Met) can increase the growth potential of hybrid turkeys and limit metabolic disorders that weaken immune function. The experiment was carried out in a full rearing cycle, from 1 to 16 wk of age, in a two-factorial randomized design with 3 levels of Arg and 2 levels of Met (90, 100 and 110% of Arg, and 30 or 45% of Met, relative to the content of dietary Lys), with 6 groups of 8 replicates per group and 18 turkeys per replicate. In the first and second month of rearing, a significant dietary Arg-by-Met interaction was noted for daily feed intake and body weight gain, and a more beneficial effect was exerted by higher Met content and medium Arg content. Throughout the experiment, the higher dietary Met level increased the final body weight (BW) of turkeys (P = 0.001). Different dietary Arg levels had no influence on the growth performance of turkeys, but the lowest level decreased dressing yield (P = 0.001), and the highest level increased the percentage of breast muscles in the final BW of turkeys (P = 0.003). The lowest Arg level (90% of Lys content) undesirably increased the concentration of the proinflammatory cytokine IL-6 (P = 0.028) and decreased globulin concentration (P = 0.001) in the blood plasma of turkeys. The higher dietary Met level (45% of Lys content) increased plasma albumin concentration (P = 0.016). It can be concluded that higher dietary levels of Met (45 vs. 30% of Lys content) and Arg (100 and 110 vs. 90% of Lys content) have a more beneficial effect on the growth performance and immune status of turkeys., (Copyright © 2019. Published by Elsevier Inc.)

y+LAT1 (encoded by SLC7A7), together with y+LAT2 (encoded by SLC7A6), is the alternative light subunits composing the heterodimeric transport system y+L for cationic and neutral amino acids. SLC7A7 mutations cause lysinuric protein intolerance (LPI), an inherited multisystem disease characterized by low plasma levels of arginine and lysine, protein-rich food intolerance, failure to thrive, hepatosplenomegaly, osteoporosis, lung involvement, kidney failure, haematologic and immunological disorders. The reason for the heterogeneity of LPI symptoms is thus far only poorly understood. Here, we aimed to quantitatively compare the expression of SLC7A7 and SLC7A6 among different human cell types and evaluate y+LAT1 and y+LAT2 contribution to arginine transport. We demonstrate that system y+L-mediated arginine transport is mainly accounted for by y+LAT1 in monocyte-derived macrophages (MDM) and y+LAT2 in fibroblasts. The kinetic analysis of arginine transport indicates that y+LAT1 and y+LAT2 share a comparable affinity for the substrate. Differences have been highlighted in the expression of SLC7A6 and SLC7A7 mRNA among different cell models: while SLC7A6 is almost equally expressed, SLC7A7 is particularly abundant in MDM, intestinal Caco-2 cells and human renal proximal tubular epithelial cells (HRPTEpC). The characterization of arginine uptake demonstrates that system y+L is operative in renal cells and in Caco-2 where, at the basolateral side, it mediates arginine efflux in exchange with leucine plus sodium. These findings explain the defective absorption/reabsorption of arginine in LPI. Moreover, y+LAT1 is the prevailing transporter in MDM sustaining a pivotal role in the pathogenesis of immunological complications associated with the disease., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Background: Bone mineral density (BMD) measurements are widely used to assess fracture risk. However, the finding that some fracture patients had high BMD together with the low contribution of drugs to osteoporosis suggests that bone strength factors other than BMD contribute to bone quality. We evaluated the amount of advanced glycation end products (AGEs) by non-invasive assays of serum and urine as well as by skin autofluorescence to measure the levels of a representative AGE, pentosidine, to investigate whether pentosidine can serve as an indirect indicator of AGEs formation in bone collagen., Methods: A total of 100 spinal surgery patients without fragility fracture (54 males and 46 females) treated at our hospital were enrolled. The amount of pentosidine in blood, urine, skin and bone (lumbar lamina) samples from these patients was measured. AGE accumulation was assessed by measuring skin autofluorescence. We examined the correlation between pentosidine content in tissues and body fluid, as well as skin AGEs with age, height, body weight, BMI, and estimated glomerular filtration rate (eGFR)., Results: A significant age-related increase in pentosidine levels in tissues was observed, while there was a significant negative correlation between tissue pentosidine and eGFR. The amount of skin pentosidine was significantly and positively correlated with pentosidine content of the bone in those under 50 years of age. Urine pentosidine also correlated positively with bone pentosidine and skin pentosidine, but only in females. The total amount of AGEs in skin did not correlate with bone pentosidine., Conclusion: In this study, the strong correlation between the pentosidine content in each sample and eGFR may indicate that renal dysfunction with advancing age increases oxidative stress and induces AGEs formation in collagen-containing tissues. The correlation of skin pentosidine concentration and eGFR, with AGEs formation in bone collagen suggests that pentosidine would be a useful indirect index of decreased bone quality. Skin AGEs estimated by autofluorescence in clinical situations may not be suitable as an indirect assessment of bone quality. Because urine pentosidine correlated positively with bone pentosidine and skin pentosidine in females, urine pentosidine may be a candidate for an indirect assessment of bone quality.

Objective: Pentosidine (PEN), a well-defined advanced glycation end product (AGE), may be affected by psychological status, given the recent findings regarding AGE receptor functions. Because AGEs can be a factor in aging and in the development or worsening of many degenerative diseases, it is important to find a way to reduce the PEN levels in our body. This study aims to investigate novel factors associated with PEN levels., Methods: A cross-sectional study involving 106 female participants (aged 59-69) was conducted in a tea-producing district in Japan. The serum concentration of PEN was detected and evaluated in relation to the participants' psychological status, which was assessed using the Japanese version of the 28-item General Health Questionnaire (GHQ) and lifestyle factors. Factors that were significantly associated with PEN were analysed using multiple linear regression analyses. Significance was defined as p < .05., Results: The serum PEN concentrations were significantly and positively associated with the total GHQ scores and BMI after controlling for covariates (standardised beta coefficient (B) = 0.26, p < .01; B = 0.27, p < .01, respectively). In addition, the PEN levels were significantly lower in participants who consumed seven cups or more of green tea per day than those who consumed six or fewer cups per day (B = 0.19, p < .05)., Conclusions: Low GHQ scores (i.e. better psychological well-being) and green tea consumption may be helpful in decreasing AGEs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aim: Advanced glycation end-products (AGEs) have been involved in the atherogenic process in the high-risk population. The goal of this study was to demonstrate that AGEs are related to subclinical atheromatous disease in subjects with low to moderate vascular risk., Methods: A cross-sectional study in which 2,568 non-diabetic subjects of both sexes without cardiovascular disease were included. Subcutaneous content of AGEs was assessed by skin autofluorescence (SAF) and subclinical atheromatous disease was measured by assessing the atheromatous plaque burden in carotid and femoral regions using ultrasonography. In addition, serum pentosidine, carboxymethyl-lysine (CML) and AGE receptors (RAGE) were assessed in a nested case-control study with 41 subjects without plaque and 41 individuals subjects with generalized disease., Results: Patients with atheromatous plaque had a higher SAF than those with no plaque (1.9 [1.7 to 2.3] vs. 1.8 [1.6 to 2.1] arbitrary units (AU), p＜0.001). The SAF correlated with the total number of affected regions (r= 0.171, p＜0.001), increasing progressively from 1.8 [1.6 to 2.1] AU in those without atheromatous disease to 2.3 [1.9 to 2.7] AU in patients with ≥ 8 plaques (p＜0.001). A correlation was also observed between SAF and the total plaque area (r=0.113, p＜0.001). The area under the Receiver Operating Characteristic curve was 0.65 (0.61 to 0.68) for identifying male subjects with atheromatous disease. The multivariable logistic regression model showed a significant and independent association between SAF and the presence of atheromatous disease. However, no significant differences in serum pentosidine, CML, and RAGE were observed., Conclusions: Increased subcutaneous content of AGEs is associated with augmented atheromatous plaque burden. Our results suggest that SAF may provide clinically relevant information to the current strategies for the evaluation of cardiovascular risk, especially among the male population.

Initial intimate apposition between implant fixtures and host bone at the surgical site is a critical factor for osseointegration of dental implants. The advanced glycation end products accumulated in the jaw bone could lead to potential failure of a dental implant during the initial integration stage, because of the inferior bone mechanical property associated with the abnormal collagen cross-linking at the material level. Here, we demonstrate the lowered creep deformation resistance and reduced dimensional recovery of jaw bone in line with high levels of pentosidine accumulation in the bone matrix which likely correlate with the pentosidine level in blood plasma. Peripheral blood samples and cortical bone samples at the surgical site were obtained from patients scheduled for dental implants in the mandible. The pentosidine levels in blood plasma were assessed. Subsequently, the relative pentosidine levels and the mechanical properties of the jaw bone were quantified by Raman microspectroscopy and nanoindentation, respectively. The nanoindentation tests revealed less creep deformation resistance and reduced time-dependent dimensional recovery of bone samples with the increase in the relative pentosidine level in the bone matrix. Higher tan δ values at the various frequencies during the dynamic indentation tests also suggested that viscoelasticity is associated with the relative intensity of pentosidine in the jaw bone matrix. We found a positive correlation between the pentosidine levels in blood plasma and the bone matrix, which in turn reduced the mechanical property of the jaw bone at the material level. Increased creep and reduced dimensional recovery of the jaw bone may diminish the mechanical interlocking of dental implants during the initial integration stage. Given the likely correlation between the plasma pentosidine level and the mechanical properties of bone, measurement of the plasma pentosidine level could serve as a new index to assess jaw bone matrix quality in advance of implant surgery., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure N ω -(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues. When various protein samples were incubated with glucose, the CMA content, detected using a monoclonal antibody, increased in a time-dependent manner only in glycated collagen, whereas the formation of N ε -(carboxymethyl)lysine (CML), a major antigenic AGE, was detected in all glycated proteins. Dominant CMA formation in glycated collagen was also observed by electrospray ionization-liquid chromatography-tandem mass spectrometry (LC-MS/MS). During incubation of glucose with collagen, CMA formation was enhanced with increasing glucose concentration, whereas it was inhibited in the presence of dicarbonyl-trapping reagents and a metal chelator. CMA formation was also observed upon incubating collagen with glyoxal, and CMA was generated in a time-dependent manner when glyoxal was incubated with type I-IV collagens. To identify hotspots of CMA formation, tryptic digests of glycated collagen were applied to an affinity column conjugated with anti-CMA. Several CMA peptides that are important for recognition by integrins were detected by LC-MS/MS and amino acid sequence analyses. CMA formation on each sequence was confirmed by incubation of the synthesized peptides with glyoxal and ribose. LC-MS detected CMA in the mouse skin at a higher level than other AGEs. Furthermore, CMA accumulation was greater in the human aorta of older individuals. Overall, our study provides evidence that CMA is a representative AGE structure that serves as a useful index to reflect the oxidation and glycation of collagen., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2019 Sho Kinoshita et al.)

Chemical cross-linking of proteins coupled with mass spectrometry analysis (CXMS) is widely used to study protein-protein interactions (PPI), protein structures, and even protein dynamics. However, structural information provided by CXMS is still limited, partly because most CXMS experiments use lysine-lysine (K-K) cross-linkers. Although superb in selectivity and reactivity, they are ineffective for lysine deficient regions. Herein, we develop aromatic glyoxal cross-linkers (ArGOs) for arginine-arginine (R-R) cross-linking and the lysine-arginine (K-R) cross-linker KArGO. The R-R or K-R cross-links generated by ArGO or KArGO fit well with protein crystal structures and provide information not attainable by K-K cross-links. KArGO, in particular, is highly valuable for CXMS, with robust performance on a variety of samples including a kinase and two multi-protein complexes. In the case of the CNGP complex, KArGO cross-links covered as much of the PPI interface as R-R and K-K cross-links combined and improved the accuracy of Rosetta docking substantially.

l-Homoarginine (hArg) is biosynthesized from l-arginine (Arg) and l-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: It has been shown that the oral aminobisphosphonate sodium alendronate (ALN) therapy reduces the risk of main fractures in osteoporotic women, but its effect on the jaw bones is poorly known. Here, we hypothesized that ALN affects the newly formed alveolar bone, particularly the quality of the type I collagen cross-linking., Methods: Osteoporosis was induced by ovariectomy (OVX) in 6-month old rabbits. Six weeks following surgery, eight animals were treated by oral gavage with ALN (OVX + ALN) and ten received placebo (OVX + Pbo). Another six rabbits which were sham operated also received placebo (SHAM + Pbo). One month following the beginning of treatment, the upper and lower left first premolars were removed. Six weeks later, the upper and the lower right first premolars were also extracted. One month after the second extraction, biopsies were collected from the maxillary extraction sites and collagen crosslinks were analyzed in the newly formed bone tissue by HPLC. Also, at this time, mandibular bone segments were subjected to μCT., Results: Animals treated with ALN achieved a roughly 2-time greater bone volume fraction value at a late healing period than animals in the other groups (p < 0.05). Collagen mean results were 2- to 4-times superior in the OVX + ALN group than in the control groups (p < 0.05). ALN-treated animals presented higher amounts of the non-enzymatic collagen cross-link pentosidine (PEN) than the sham-operated rabbits (p < 0.05), whereas the OVX + Pbo group presented the highest amount of PEN (p < 0.05)., Conclusion: Alendronate increases bone volume and collagen accumulation, but does not fully rescue the non-osteoporotic alveolar tissue quality as is evident from the increased quantity of pentosidine., (Copyright © 2018. Published by Elsevier Inc.)

Although polyubiquitin chains linked through all lysines of ubiquitin exist, specific functions are well-established only for lysine-48 and lysine-63 linkages in Saccharomyces cerevisiae . To uncover pathways regulated by distinct linkages, genetic interactions between a gene deletion library and a panel of lysine-to-arginine ubiquitin mutants were systematically identified. The K11R mutant had strong genetic interactions with threonine biosynthetic genes. Consistently, we found that K11R mutants import threonine poorly. The K11R mutant also exhibited a strong genetic interaction with a subunit of the anaphase-promoting complex (APC), suggesting a role in cell cycle regulation. K11-linkages are important for vertebrate APC function, but this was not previously described in yeast. We show that the yeast APC also modifies substrates with K11-linkages in vitro, and that those chains contribute to normal APC-substrate turnover in vivo. This study reveals comprehensive genetic interactomes of polyubiquitin chains and characterizes the role of K11-chains in two biological pathways., Competing Interests: FM, AM, AD, HB, JJ, JX, MS, MN, RT, CP, LS, NK, DT No competing interests declared, DS is affiliated with Amgen Research. The author has no other competing interests to declare., (© 2018, Meza Gutierrez et al.)

Sirtuins (SIRTs) are a class of lysine deacylases that regulate cellular metabolism and energy homeostasis. Although sirtuins have been proposed to function in nutrient sensing and signaling, the underlying mechanism remains elusive. SIRT7, a histone H3K18-specific deacetylase, epigenetically controls mitochondria biogenesis, ribosomal biosynthesis, and DNA repair. Here, we report that SIRT7 is methylated at arginine 388 (R388), which inhibits its H3K18 deacetylase activity. Protein arginine methyltransferase 6 (PRMT6) directly interacts with and methylates SIRT7 at R388 in vitro and in vivo R388 methylation suppresses the H3K18 deacetylase activity of SIRT7 without modulating its subcellular localization. PRMT6-induced H3K18 hyperacetylation at SIRT7-target gene promoter epigenetically promotes mitochondria biogenesis and maintains mitochondria respiration. Moreover, high glucose enhances R388 methylation in mouse fibroblasts and liver tissue. PRMT6 signals glucose availability to SIRT7 in an AMPK-dependent manner. AMPK induces R388 hypomethylation by disrupting the association between PRMT6 and SIRT7. Together, PRMT6-induced arginine methylation of SIRT7 coordinates glucose availability with mitochondria biogenesis to maintain energy homeostasis. Our study uncovers the regulatory role of SIRT7 arginine methylation in glucose sensing and mitochondria biogenesis., (© 2018 The Authors.)

In recent decades, several methods based on biochemical and molecular changes caused by aging have been proposed to improve the accuracy of forensic age estimation. The present study aimed to measure changes in furosine and pentosidine, two markers of non-enzymatic glycation of proteins (NEGs), in human dentine and clavicle with aging, and to identify possible differences between turnover rates in different mineralized tissues. Furosine and pentosidine were quantified in 32 dentine samples from living donors between 14 and 80 years of age, and in a second group of samples consisting of a tooth and a piece of clavicle collected from the same cadaver (15 individuals aged 18 to 85 years). Furosine concentration was much higher than pentosidine concentration in the same tissue, although they were strongly correlated in both dentine and bone. A close relationship between furosine and/or pentosidine content and chronological age was found in both tissues (r &gt; 0.93). Moreover, age estimation was more accurate when furosine or pentosidine content was determined in dentine, with specificity values for the tests higher than 82% in all age groups. In clavicle, furosine concentration and pentosidine concentration were much lower (2.6-fold and 3.1-fold, respectively) than in dentine from the same individuals. In conclusion, although the results show strong correlations between chronological age and furosine or pentosidine concentrations determined in mineralized tissues, there is still a need for further research with larger data sets, including patients with diabetes.

Background: Airflow limitation in COPD is caused by a mixture of small airways obstruction and alveolar destruction., Objective: To evaluate the contributions of these factors to airflow limitation through measurement of two biomarkers, pentosidine and vascular endothelial growth factor (VEGF), which reflect pathology or function of the lower respiratory tract of COPD., Methods: We measured pentosidine and VEGF levels in induced sputum from 23 non-smokers, 26 smokers without COPD, and 43 smokers with COPD. We evaluated the correlations of two biomarkers levels with the grade of low attenuation area (LAA) in high-resolution computed tomographic scans and the Δ N 2 from the nitrogen washout curve., Results: Pentosidine levels were significantly higher in smokers with COPD than in non-smokers and smokers without COPD. In contrast, VEGF levels were significantly lower in smokers without COPD than in non-smokers, and further decreased in smokers with COPD. In the four-stage classification of LAA grading, pentosidine levels steeply increased from grade I to Ⅳ, while VEGF levels decreased with increasing severity of LAA grade. Pentosidine levels were positively correlated with Δ N 2 in COPD patients with mild emphysema. In contrast, VEGF levels were inversely correlated with Δ N 2 in COPD patients with severe emphysema., Conclusion: Pentosidine level is responsible for the severity of small airways obstruction, while VEGF level reflects the magnitude of alveolar destruction. Thus, simultaneous measurement of pentosidine and VEGF levels may be a promising approach to discriminate the severity of small airways obstruction and alveolar destruction in the lower respiratory tract of COPD., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chronic inflammation and Advanced Glycation End products (AGE) are associated with sarcopenia. Decreased voluntary muscle activation and increased antagonist coactivation can contribute to age-related muscle weakness. The influence of chronic inflammation and AGE in these neuromuscular mechanisms is not clear. We studied whether a relation exists between circulating levels of inflammatory cytokines and AGEs as well as the interplay between agonist and antagonist muscle activation. We studied 64 community-dwelling old subjects, during a maximal isometric voluntary contraction (MVC) and a reaction-time (RT) test of the upper limb. Twenty-five circulating inflammatory biomarkers were determined. Linear regression showed significant relationships between chronic inflammation and six muscle activation parameters. MIP-1β showed a significant negative relation with antagonist coactivation (during MVC) and antagonist muscle activity during pre-movement time (PMT) and movement time (MT) (during RT). A higher level of pentosidine (AGE) was predictive for a longer PMT. We conclude that in older relatively healthy persons antagonist muscle activation is influenced by chronic inflammation, contributing to age-related muscle weakness. Our results also suggest a mechanical and inflammatory influence of pentosidine in upper limb slowing of movement. These findings show novel insight in underlying mechanisms of age-related muscle weakness., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background Information: Repair of damaged DNA is essential for maintaining genomic stability. TP53-binding protein 1 (53BP1) plays an important role in repair of the DNA double-strand breaks. Nuclear localisation of 53BP1 depends on importin β and nucleoporin 153, but the type and location of 53BP1 nuclear localisation signal (NLS) have yet to be determined., Results: Here, we show that nuclear import of 53BP1 depends on two basic regions, namely 1667-KRK-1669 and 1681-KRGRK-1685, which are both needed for importin binding. Lysine 1667 is essential for interaction with importin and its substitution to arginine reduced nuclear localisation of 53BP1. Furthermore, we have found that CDK1-dependent phosphorylation of 53BP1 at S1678 impairs importin binding during mitosis. Phosphorylation-mimicking mutant S1678D showed reduced nuclear localisation, suggesting that phosphorylation of the NLS interferes with nuclear import of the 53BP1 CONCLUSIONS: We show that 53BP1 contains a classical bipartite NLS 1666-GKRKLITSEEERSPAKRGRKS-1686, which enables the importin-mediated nuclear transport of 53BP1. Additionally, we found that posttranslational modification within the NLS region can regulate 53BP1 nuclear import., Significance: Our results indicate that integrity of the NLS is important for 53BP1 nuclear localisation. Precise mapping of the NLS will facilitate further studies on the effect of posttranslational modifications and somatic mutations on the nuclear localisation 53BP1 and DNA repair., (© 2018 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

Ageing of the human organism results in the accumulation of modified molecules. Some of these molecular changes may be used for age estimation, as already shown for aspartic acid racemization (AAR). Another example for an accumulation of damaged molecules is advanced glycation end products (AGEs). We examined, (1) if the correlation between the concentration of AGEs (pentosidine) in root dentine and age is close enough to be used as basis for age estimation, and (2) if the combined analysis of AGEs and AAR in dentine may be a useful approach to rule out or to detect relevant effects of confounding factors in age estimation. We determined the pentosidine content of root dentine samples of 64 healthy teeth as well as in carious, "pink", diabetic and heated teeth, and in teeth after different storage times. In 23 teeth, the extent of aspartic acid racemization (AAR) was determined in parallel. We observed a close relationship between the concentration of pentosidine in dentine and chronological age (r = 0.94) in healthy teeth. The analysis of pentosidine in dentine can theoretically be used as a basis for age estimation in healthy teeth of non-diabetic individuals; diabetic individuals may exhibit very high pentosidine levels in dentine. This finding limits the application of this method, since information regarding the question if an unidentified person suffered from diabetes mellitus or not are missing in most cases. Moreover, the method is not suitable to identify or rule out the influence of confounding factors in age estimation based on AAR, since both methods are sensible to the most relevant confounding factors (caries, heat).

Oxygenase-catalysed post-translational modifications of basic protein residues, including lysyl hydroxylations and N ε -methyl lysyl demethylations, have important cellular roles. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), which genetic studies reveal is essential in animal development, is reported as a histone N ε -methyl lysine demethylase (KDM). Here we report how extensive screening with peptides based on JMJD5 interacting proteins led to the finding that JMJD5 catalyses stereoselective C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6). High-resolution crystallographic analyses reveal overall fold, active site and substrate binding/product release features supporting the assignment of JMJD5 as an arginine hydroxylase rather than a KDM. The results will be useful in the development of selective oxygenase inhibitors for the treatment of cancer and genetic diseases.

Accumulation of advanced glycation end products (AGEs) is accompanied by increased free radical activity which contributes to ageing and the development or worsening of degenerative diseases. Apart from other physiological factors, AGEs are also an important biomarker for premature ageing. Here we report protein modifications (glycation) in a mouse model of lamin B receptor deficient ic J /ic J mice displaying skin defects similar to those of classical progeria. Therefore, we analysed AGE-modifications in protein extracts from various tissues of ic J /ic J mice. Our results demonstrated that pentosidine as well as argpyrimidine were increased in ic J /ic J mice indicating a modification specific increase in biomarkers of ageing, especially derived from glycolysis dependent methylglyoxal. Furthermore, the expression of AGE-preventing enzymes (Glo1, Fn3k) differed between ic J /ic J and control mice. The results indicate that not only lamin A but also the lamin B receptor may be involved in ageing processes.

The heparin-binding glycoprotein YKL-40 (CHI3L1) is intimately associated with microvascularization in multiple human diseases including cancer and inflammation. However, the heparin-binding domain(s) pertinent to the angiogenic activity have yet been identified. YKL-40 harbors a consensus heparin-binding motif that consists of positively charged arginine (R) and lysine (K) (RRDK; residues 144-147); but they don't bind to heparin. Intriguingly, we identified a separate KR-rich domain (residues 334-345) that does display strong heparin binding affinity. A short synthetic peptide spanning this KR-rich domain successfully competed with YKL-40 and blocked its ability to bind heparin. Three individual point mutations, where alanine (A) substituted for K or R (K337A, K342A, R344A), led to remarkable decreases in heparin-binding ability and angiogenic activity. In addition, a neutralizing anti-YKL-40 antibody that targets these residues and prevents heparin binding impeded angiogenesis in vitro. MDA-MB-231 breast cancer cells engineered to express ectopic K337A, K342A or R344A mutants displayed reduced tumor development and compromised tumor vessel formation in mice relative to control cells expressing wild-type YKL-40. These data reveal that the KR-rich heparin-binding motif is the functional heparin-binding domain of YKL-40. Our findings shed light on novel molecular mechanisms underlying endothelial cell angiogenesis promoted by YKL-40 in a variety of diseases., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Although the endogenous secretory receptor for advanced glycation end products (esRAGE) has been associated with reduced activity of pentosidine (PEN), the association between PEN, esRAGE, and fracture is poorly understood., Objectives: To evaluate the ability of serum PEN and esRAGE levels to predict fragility fractures., Methods: A cohort of 1285 Japanese men aged ≥65 years old participated in a 2007 to 2008 Fujiwara-kyo Osteoporosis Risk in Men study baseline survey, as part of the Fujiwara-kyo prospective cohort study. Those participants provided information regarding any fractures they experienced during 5 years. The baseline bone mineral density (BMD) was measured. Hazard ratios (HRs) per one standard deviation increase of log-transformed serum levels of PEN, esRAGE, and esRAGE-to-PEN ratio were estimated at baseline., Results: Twenty-five participating men suffered incident clinical fragility fractures. The crude HRs (95% confidence interval) for PEN, esRAGE, and esRAGE-to-PEN ratio were 1.56 (1.05 to 2.31), 0.79 (0.54 to 1.15), and 0.65 (0.44 to 0.95), respectively. HRs for PEN adjusted for age, esRAGE, and T score of BMD at femoral neck (FN) and lumbar spine (LS) were 1.48 (1.00 to 2.18) and 1.51 (1.03 to 2.21), respectively. The marginal significance adjusted for BMD at FN and the statistical significance adjusted for BMD at LS were attenuated after additional adjustment for glycated hemoglobin A1c level (P = 0.111 and 0.072, respectively). The HRs for esRAGE-to-PEN ratio adjusted for age, glycated hemoglobin A1c, and T-score of BMD at FN and LS were 0.67 (0.45 to 0.98) and 0.64 (0.43 to 0.95)., Conclusions: Higher esRAGE-to-PEN ratios were associated with decreased risk of fragility fractures independent of BMD among elderly Japanese men., (Copyright © 2017 Endocrine Society)

Extensive research has linked the amyloid-beta (Aβ) peptide to neurological dysfunction in Alzheimer's disease (AD). Insoluble Aβ plaques in the AD patient brain contain high concentrations of advanced glycation end-products (AGEs) as well as transition metal ions. This research elucidated the roles of Aβ, sugars, and Cu 2+ in the oxidative stress mechanism of AD at the molecular level. Mass spectral (MS) analysis of the reactions of Aβ with two representative sugars, ribose-5-phosphate (R5P) and methylglyoxal (MG), revealed Lys-16 and Arg-5 as the primary glycation sites. Quantitative analysis of superoxide [Formula: see text] production by a cyt c assay showed that Lys-16 generated four times as much [Formula: see text] as Arg-5. Lys-16 and Arg-5 in Aβ 1-40 are both adjacent to histidine residues, which are suggested to catalyze glycation. Additionally, Lys-16 is close to the central hydrophobic core (Leu-17-Ala-21) and to His-13, both of which are known to lower the pKa of the residue, leading to increased deprotonation of the amine and an enhanced glycation reactivity compared to Arg-5. Gel electrophoresis results indicated that all three components of AD plaques-Aβ 1-40 , sugars, and Cu 2+ -are necessary for DNA damage. It is concluded that the glycation of Aβ 1-40 with sugars generates significant amounts of [Formula: see text], owing to the rapid glycation of Lys-16 and Arg-5. In the presence of Cu 2+ , [Formula: see text] converts to hydroxyl radical (HO·), the source of oxidative stress in AD.

Chemical modifications of the DNA and nucleosomal histones tightly control the gene transcription program in eukaryotic cells. The "histone code" hypothesis proposes that the frequency, combination, and location of post-translational modifications (PTMs) of the core histones compose a complex network of epigenetic regulation. Currently, there are at least 23 different types and >450 histone PTMs that have been discovered, and the PTMs of lysine and arginine residues account for a crucial part of the histone code. Although significant progress has been achieved in recent years, the molecular basis for the histone code is far from being fully understood. In this study, we investigated how naturally occurring N-terminal acetylation and PTMs of histone H4 lysine-5 (H4K5) affect arginine-3 methylation catalyzed by both type I and type II PRMTs at the biochemical level. Our studies found that acylations of H4K5 resulted in decreased levels of arginine methylation by PRMT1, PRMT3, and PRMT8. In contrast, PRMT5 exhibits an increased rate of arginine methylation upon H4K5 acetylation, propionylation, and crotonylation, but not upon H4K5 methylation, butyrylation, or 2-hydroxyisobutyrylation. Methylation of H4K5 did not affect arginine methylation by PRMT1 or PRMT5. There was a small increase in the rate of arginine methylation by PRMT8. Strikingly, a marked increase in the rate of arginine methylation was observed for PRMT3. Finally, N-terminal acetylation reduced the rate of arginine methylation by PRMT3 but had little influence on PRMT1, -5, and -8 activity. These results together highlight the underlying mechanistic differences in substrate recognition among different PRMTs and pave the way for the elucidation of the complex interplay of histone modifications.

Objectives: Non-enzymatic glycation is closely associated with altered mechanical properties of connective tissue. Pregnancy, marked with high levels of female hormones, confers unique alteration to the mechanical properties of pelvic connective tissues in order to meet their physiological demands. However, there are few studies on glycation content and its influence on the mechanical properties of pelvic connective tissues during pregnancy. We hypothesise that the glycation content in pelvic tissues will change with a corresponding alteration in their mechanical properties, and that these changes are influenced by hormone levels. This study aims to investigate the correlation of vaginal tissue glycation content and mechanical property changes during pregnancy in association with the expression of a key pregnancy hormone (oestrogen) receptor, and an antioxidant enzyme, glyoxalase I., Study Design: A rat vaginal tissue model (tissues from non-pregnant and E15-E18 (last trimester) pregnant rats) was used in this study. Mechanical characteristics of vaginal tissues were analysed by a ball-indentation technique while modulus and morphology of the collagen fibrils within the tissues were measured with atomic force microscopy. A glycation marker, pentosidine, was quantified by a high performance liquid chromatography. The expression of oestrogen receptor and glyoxalase I in the tissue was qualified by immunochemical staining. The glycosaminoglycan (GAG) concentration difference in the tissues were quantified by a biochemical assay., Results: Pregnant rat vaginal tissue was characterised by significantly lower amounts of pentosidine, higher oestrogen receptor and glyoxalase I expression with larger creep, lower elastic modulus, larger fibril diameter and higher GAG content than their non-pregnant counterpart. There was a negative correlation between pentosidine and vaginal tissue creep., Conclusion: There was a reduction in vaginal tissue pentosidine in pregnancy with an associated increase in oestrogen receptor and glyoxalase I immunoexpression. Reduced glycation was associated with increased creeping of vaginal tissue. Oestrogen may therefore play a role in the increase of the vaginal wall's capacity to stretch through glyoxalase I up-regulation and subsequent glycation reduction. The new insight of the correlation of women's oestrogen level, glycation reaction and pelvic tissue mechanical property from this study may enhance our understanding of some pelvic organ diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Stable isotope labeling by amino acids in cell culture (SILAC) enables the relative quantification of protein amounts and posttranslational modifications in complex biological samples through the use of stable heavy isotope-labeled amino acids. Here we describe methods for the application of SILAC to fission yeast Schizosaccharomyces pombe using either labeled lysine or a combination of labeled lysine and labeled arginine. The latter approach is more complicated than the use of labeled lysine alone but may yield a more comprehensive (phospho)proteomic analysis. The protocol includes methods for construction of SILAC-compatible strains, growth of cultures in labeled medium, cell harvesting, and protein extraction., (© 2017 Cold Spring Harbor Laboratory Press.)

Purpose: To explore factors associated with pentosidine accumulation in the human vitreous., Methods: Vitreous samples were obtained during trans pars plana vitrectomy for macular hole or rhegmatogenous retinal detachment. Patient characteristics included age, gender, and diabetes mellitus. Ocular characteristics included pseudophakia, posterior vitreous detachment, and presence of intraocular fibrosis (epiretinal membrane, proliferative vitreoretinopathy, or both). Pentosidine concentration as a measure of accumulation of advanced glycation end products was determined by high performance liquid chromatography., Results: Pentosidine concentrations were measured in 222 vitrectomy samples (118 female and 104 male patients [median age 66 years], treated for macular hole [n = 105] or rhegmatogenous retinal detachment [n = 117]). Pentosidine was found to accumulate significantly with age (P < 0.001). After correction for age, a multivariable linear regression model revealed significantly higher pentosidine values in eyes with intraocular fibrosis (P = 0.001), in phakic as compared with pseudophakic eyes (P = 0.02), and in the absence of a complete posterior vitreous detachment (P = 0.018). The authors found no association with diabetes mellitus or gender., Conclusion: This study confirmed an age-related pentosidine accumulation in the vitreous and found new factors relating to pentosidine levels. Findings support the hypothesis of enzyme-induced vitreous liquefaction and the hypothesis of pentosidine as a pro-fibrotic factor.

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei., Competing Interests: The authors have declared that no competing interests exist.

The mechanistic Target of Rapamycin Complex 1 (mTORC1) is a major regulator of eukaryotic growth that coordinates anabolic and catabolic cellular processes with inputs such as growth factors and nutrients, including amino acids. In mammals arginine is particularly important, promoting diverse physiological effects such as immune cell activation, insulin secretion, and muscle growth, largely mediated through activation of mTORC1 (refs 4, 5, 6, 7). Arginine activates mTORC1 upstream of the Rag family of GTPases, through either the lysosomal amino acid transporter SLC38A9 or the GATOR2-interacting Cellular Arginine Sensor for mTORC1 (CASTOR1). However, the mechanism by which the mTORC1 pathway detects and transmits this arginine signal has been elusive. Here, we present the 1.8 Å crystal structure of arginine-bound CASTOR1. Homodimeric CASTOR1 binds arginine at the interface of two Aspartate kinase, Chorismate mutase, TyrA (ACT) domains, enabling allosteric control of the adjacent GATOR2-binding site to trigger dissociation from GATOR2 and downstream activation of mTORC1. Our data reveal that CASTOR1 shares substantial structural homology with the lysine-binding regulatory domain of prokaryotic aspartate kinases, suggesting that the mTORC1 pathway exploited an ancient, amino-acid-dependent allosteric mechanism to acquire arginine sensitivity. Together, these results establish a structural basis for arginine sensing by the mTORC1 pathway and provide insights into the evolution of a mammalian nutrient sensor.

Histone PTMs play a crucial role in regulating chromatin structure and function, with impact on gene expression. MS is nowadays widely applied to study histone PTMs systematically. Because histones are rich in arginine and lysine, classical shot-gun approaches based on trypsin digestion are typically not employed for histone modifications mapping. Instead, different protocols of chemical derivatization of lysines in combination with trypsin have been implemented to obtain "Arg-C like" digestion products that are more suitable for LC-MS/MS analysis. Although widespread, these strategies have been recently described to cause various side reactions that result in chemical modifications prone to be misinterpreted as native histone marks. These artefacts can also interfere with the quantification process, causing errors in histone PTMs profiling. The work of Paternoster V. et al. is a quantitative assessment of methyl-esterification and other side reactions occurring on histones after chemical derivatization of lysines with propionic anhydride [Proteomics 2016, 16, 2059-2063]. The authors estimate the effect of different solvents, incubation times, and pH on the extent of these side reactions. The results collected indicate that the replacement of methanol with isopropanol or ACN not only blocks methyl-esterification, but also significantly reduces other undesired unspecific reactions. Carefully titrating the pH after propionic anhydride addition is another way to keep methyl-esterification under control. Overall, the authors describe a set of experimental conditions that allow reducing the generation of various artefacts during histone propionylation., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Escherichia coli DedA/Tvp38 family proteins YghB and YqjA are putative membrane transporters with 62% amino acid identity and overlapping functions. An E. coli strain (BC202) with nonpolar ΔyghB and ΔyqjA mutations displays cell-division defects and temperature sensitivity and is sensitive to antibiotics and alkaline pH. In this study, we performed site-directed mutagenesis on conserved, charged amino acids of YqjA and YghB. We discovered two conserved predicted membrane-embedded arginines (R130 and R136) that are critical for function in both proteins as defined by their ability to complement BC202 phenotypes, when expressed from a plasmid. Lysine can substitute for arginine at position R130 indicating a charge dependence at this position, but could not substitute at R136. In light of the established role that arginine plays in the translocation mechanism of numerous membrane transporters, we hypothesize that these amino acids play a role in the transport mechanism of these DedA/Tvp38 family proteins., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Interactions between polyelectrolytes and oppositely charged surfactants have been in a great interest for several decades, yet the conventional surfactants may cause a problem in medical applications. Interactivity between polysaccharide hyaluronan (HA) and amino acids Lysine, 6-Aminocaproic acid (6-AcA), and Arginine as an alternative system is reported. The interactions were investigated by means of rheology and electric conductance and the electronic structures were explored by the density functional theory (DFT). Lysine exhibits the strongest interaction of all, which was manifested, e.g. by nearly 6-time drop of the initial viscosity comparing with only 1.3-time lower value in the case of 6-AcA. Arginine interaction with HA was surprisingly weaker in terms of viscosity than that of Lysine due to a lower and delocalized charge density on its guanidine group. According to the DFT calculations, the binding of Lysine to HA was found to be more flexible, while Arginine creates more rigid structure with HA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Cysteamine is used to treat cystinosis via the modification of cysteine residues substituting arginine in mutant proteins., Objectives: We investigated the effect of cysteamine on mutant argininosuccinate lyase (ASL), the second most common defect in the urea cycle., Methods: In an established mammalian expression system, 293T cell lysates were produced after transfection with all known cysteine for arginine mutations in the ASL gene (p.Arg94Cys, p.Arg95Cys, p.Arg168Cys, p.Arg379Cys, and p.Arg385Cys), allowing testing of the effect of cysteamine over 48 h in the culture medium as well as for 1 h immediately prior to the enzyme assay., Results: Cysteamine at low concentrations showed no effect on 293T cell viability, ASL protein expression, or ASL activity when applied during cell culture. However, incubation of transfected cells with 0.05 mM cysteamine immediately before the enzyme assay resulted in increased ASL activity of p.Arg94Cys, p.Arg379Cys, and p.Arg385Cys by 64, 20, and 197 %, respectively, and this result was significant (p < 0.01). Cell lysates carrying p.Arg385Cys and treated with cysteamine recover enzyme activity that is similar to the untreated designed mutation p.Arg385Lys, providing circumstantial evidence for the assumed cysteamine-induced change of a cysteine to a lysine analogue., Conclusion: Since 12 % of all known genotypes in ASL deficiency are affected by a cysteine for arginine mutation, we conclude that the potential of cysteamine or of related substances as remedy for this disease should be investigated further.