The Molecular and Physiological Effects of Protein-Derived Polyamines in the Intestine.

Consumption of a high-protein diet increases protein entry into the colon. Colonic microbiota can ferment proteins, which results in the production of protein fermentation end-products, like polyamines. This review describes the effects of polyamines on biochemical, cellular and physiological processes, with a focus on the colon. Polyamines (mainly spermine, spermidine, putrescine and cadaverine) are involved in the regulation of protein translation and gene transcription. In this, the spermidine-derived hypusination modification of EIF5A plays an important role. In addition, polyamines regulate metabolic functions. Through hypusination of EIF5A, polyamines also regulate translation of mitochondrial proteins, thereby increasing their expression. They can also induce mitophagy through various pathways, which helps to remove damaged organelles and improves cell survival. In addition, polyamines increase mitochondrial substrate oxidation by increasing mitochondrial Ca2+-levels. Putrescine can even serve as an energy source for enterocytes in the small intestine. By regulating the formation of the mitochondrial permeability transition pore, polyamines help maintain mitochondrial membrane integrity. However, their catabolism may also reduce metabolic functions by depleting intracellular acetyl-CoA levels, or through production of toxic by-products. Lastly, polyamines support gut physiology, by supporting barrier function, inducing gut maturation and increasing longevity. Polyamines thus play many roles, and their impact is strongly tissue- and dose-dependent. However, whether diet-derived increases in colonic luminal polyamine levels also impact intestinal physiology has not been resolved yet.

Extracellular lysine 38 plays a crucial role in pH-dependent transport via human monocarboxylate transporter 1.

Human monocarboxylate transporters (hMCTs) are expressed in many tissues and mediate the transport of various substrates across the plasma membrane. Among hMCTs, hMCT1-4 cotransport H+ with monocarboxylates such as pyruvate and l-lactate, implying that these proteins recognize both substrate and H+. However, the mechanism of translocation, and particularly that of hMCT1 pH-dependent transport, remains largely unknown. This study aimed at identifying residues involved in the pH dependence of hMCT1 using a combination of amino acid-modifying reagents, site-directed mutagenesis in a Xenopus laevis oocyte expression system, and homology modeling. We showed that diethyl pyrocarbonate (DEPC), phenylglyoxal (PGO), and 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid disodium salt (DIDS), which react with histidine, arginine, and lysine residues respectively, all inhibited hMCT1 activity. Since DEPC, PGO, and DIDS are membrane impermeable reagents, we mutated to other residues individual histidine, arginine, and lysine residues located within the extracellular regions of hMCT1. Analyses of these mutants demonstrated that except for K38, the extracellular basic residues of hMCT1 were not involved in its transport activity and pH dependence. Moreover, analyses of various mutants in which K38 was substituted for another residue and of an hMCT1 homology model focusing on the location of K38 in the three-dimensional structure delineated the mechanism of hMCT1 pH dependence. Collectively, our data indicate that K38 plays an essential role in hMCT1 transport activity. We would like to propose a mechanism whereby K38 is positioned within a hydrophobic and narrow cavity that is part of the transport pathway, and regulates pH-dependent gating of hMCT1.

Hepatocyte TNF Receptor-Associated Factor 6 Aggravates Hepatic Inflammation and Fibrosis by Promoting Lysine 6-Linked Polyubiquitination of Apoptosis Signal-Regulating Kinase 1.

Activation of apoptosis signal-regulating kinase 1 (ASK1) is a key driving force of the progression of nonalcoholic steatohepatitis (NASH) and represents an attractive therapeutic target for NASH treatment. However, the molecular and cellular mechanisms underlying ASK1 activation in the pathogenesis of NASH remain incompletely understood. In this study, our data unequivocally indicated that hyperactivated ASK1 in hepatocytes is a potent inducer of hepatic stellate cell (HSC) activation by promoting the production of hepatocyte-derived factors. Our previous serial studies have shown that the ubiquitination system plays a key role in regulating ASK1 activity during NASH progression. Here, we further demonstrated that tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes lysine 6 (Lys6)-linked polyubiquitination and subsequent activation of ASK1 to trigger the release of robust proinflammatory and profibrotic factors in hepatocytes, which, in turn, drive HSC activation and hepatic fibrosis. Consistent with the in vitro findings, diet-induced liver inflammation and fibrosis were substantially attenuated in Traf6+/- mice, whereas hepatic TRAF6 overexpression exacerbated these abnormalities. Mechanistically, Lys6-linked ubiquitination of ASK1 by TRAF6 facilitates the dissociation of thioredoxin from ASK1 and N-terminal dimerization of ASK1, resulting in the boosted activation of ASK1-c-Jun N-terminal kinase 1/2 (JNK1/2)-mitogen-activated protein kinase 14(p38) signaling cascade in hepatocytes. Conclusion: These results suggest that Lys6-linked polyubiquitination of ASK1 by TRAF6 represents a mechanism underlying ASK1 activation in hepatocytes and a key driving force of proinflammatory and profibrogenic responses in NASH. Thus, inhibiting Lys6-linked polyubiquitination of ASK1 may serve as a potential therapeutic target for NASH treatment.

Lysine Fermentation: History and Genome Breeding.

Lysine fermentation by Corynebacterium glutamicum was developed in 1958 by Kyowa Hakko Kogyo Co. Ltd. (current Kyowa Hakko Bio Co. Ltd.) and is the second oldest amino acid fermentation process after glutamate fermentation. The fundamental mechanism of lysine production, discovered in the early stages of the process's history, gave birth to the concept known as "metabolic regulatory fermentation," which is now widely applied to metabolite production. After the development of rational metabolic engineering, research on lysine production first highlighted the need for engineering of the central metabolism from the viewpoints of precursor supply and NADPH regeneration. Furthermore, the existence of active export systems for amino acids was first demonstrated for lysine in C. glutamicum, and this discovery has resulted in the current recognition of such exporters as an important consideration in metabolite production. Lysine fermentation is also notable as the first process to which genomics was successfully applied to improve amino acid production. The first global "genome breeding" strategy was developed using a lysine producer as a model; this has since led to new lysine producers that are more efficient than classical industrial producers. These advances in strain development technology, combined with recent systems-level approaches, have almost achieved the optimization of entire cellular systems as cell factories for lysine production. In parallel, the continuous improvement of the process has resulted not only in fermentation processes with reduced load on downstream processing but also in commercialization of various product forms according to their intended uses. Nowadays lysine fermentation underpins a giant lysine demand of more than 2 million metric tons per year.

Molecular mechanism underlying ß1 regulation in voltage- and calcium-activated potassium (BK) channels.

Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory ß-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary ß-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between ß1 and ß3 or ß2 auxiliary subunits, we were able to identify that the cytoplasmic regions of ß1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of ß1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of ß1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the ß1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.

A hypusine-eIF5A-PEAK1 switch regulates the pathogenesis of pancreatic cancer.

Deregulation of protein synthesis is a hallmark of cancer cell proliferation, survival, and metastatic progression. eIF5A1 and its highly related isoform eIF5A2 are translation initiation factors that have been implicated in a range of human malignancies, but how they control cancer development and disease progression is still poorly understood. Here, we investigated how eIF5A proteins regulate pancreatic ductal adenocarcinoma (PDAC) pathogenesis. eIF5A proteins are the only known proteins regulated by a distinct posttranslational modification termed hypusination, which is catalyzed by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). The highly selective nature of the hypusine modification and its amenability to pharmacologic inhibition make eIF5A proteins attractive therapeutic targets. We found that the expression and hypusination of eIF5A proteins are upregulated in human PDAC tissues and in premalignant pancreatic intraepithelial neoplasia tissues isolated from Pdx-1-Cre: LSL-KRAS(G12D) mice. Knockdown of eIF5A proteins in PDAC cells inhibited their growth in vitro and orthotopic tumor growth in vivo, whereas amplification of eIF5A proteins increased PDAC cell growth and tumor formation in mice. Small-molecule inhibitors of DHPS and DOHH both suppressed eIF5A hypusination, preventing PDAC cell growth. Interestingly, we found that eIF5A proteins regulate PDAC cell growth by modulating the expression of PEAK1, a nonreceptor tyrosine kinase essential for PDAC cell growth and therapy resistance. Our findings suggest that eIF5A proteins utilize PEAK1 as a downstream effector to drive PDAC pathogenesis and that pharmacologic inhibition of the eIF5A-hypusine-PEAK1 axis may provide a novel therapeutic strategy to combat this deadly disease.

Determining the optimal isoleucine:lysine ratio for ten- to twenty-two-kilogram and twenty-four- to thirty-nine-kilogram pigs fed diets containing nonexcess levels of leucine.

Three 21-d experiments were conducted to determine the optimum standardized ileal digestible (SID) Ile:Lys ratio in 10- to 22-kg and 24- to 39-kg pigs. In Exp. 1, 144 Yorkshire pigs (initial BW = 10.2 kg) were assigned to 6 diets with 6 pens per treatment. Diets 1 to 5 were formulated to contain 5 graded SID Ile:Lys (44, 51, 57, 63, and 70%), 1.18% SID Leu, and 0.90% SID Lys (second limiting). Diet 6 (diet 5 with added Lys) was formulated (1.06% SID Lys) as a positive control. Pigs fed diet 6 had higher (P < 0.05) ADG and G:F and lower (P < 0.05) plasma urea N (PUN) than pigs fed diet 5 (P < 0.02), indicating that Lys was limiting in diets 1 to 5. Final BW, ADG, and ADFI increased (linear and quadratic, P < 0.05) while G:F and PUN at d 21 were not affected (P > 0.10) by dietary Ile:Lys. Overall, ADG and ADFI were highest for pigs fed diet 2 (51% SID Ile:Lys). In Exp. 2, 216 Yorkshire pigs (initial BW = 9.6 kg) were assigned to 9 diets with 6 pens per treatment. Diets 1 to 4 contained 0.40, 0.47, 0.54, and 0.61% SID Ile, respectively, and 1.21% SID Lys; diets 5 to 8 contained 0.72, 0.84, 0.96, and 1.08% SID Lys, respectively, and 0.68% SID Ile. Diet 9 was high in both Ile and Lys (0.68% SID Ile and 1.21% SID Lys). All diets contained 1.21% SID Leu. The ADG and G:F increased (linear and quadratic, P < 0.05) as SID Ile:Lys increased (diets 1 to 4 and 9). The ADG and G:F increased (linear, P < 0.05) as SID Lys increased (diets 5 to 9). The PUN at d 21 decreased (linear, P < 0.05) by increasing dietary Ile:Lys. The SID Ile:Lys to optimize ADG was 46% by curvilinear plateau or exponential regression. For G:F, the optimal SID Ile:Lys was 47 and 51% by curvilinear plateau and exponential regressions, respectively. In Exp. 3, 80 pigs (PIC 327 × C23; initial BW = 24.0 kg) were allotted to 5 treatments with 4 pigs per pen. Diets 1 to 5 were formulated to contain 5 graded SID Ile:Lys (39, 46, 53, 61, and 68%), 1.17% SID Leu, and 0.91% SID Lys (second limiting). Final BW and ADG increased (linear and quadratic, P < 0.05) and ADFI increased (linear, P = 0.047) as SID Ile:Lys increased. Using ADG and G:F, the optimum SID Ile:Lys was 54 and 53%, respectively, by curvilinear plateau and exponential regression. The PUN was minimized at 53 and 59% SID Ile:Lys by curvilinear plateau and broken line regression. Overall, the average optimum SID Ile:Lys was approximately 51% for 10- to 22-kg pigs and 54% for 24- to 39-kg pigs fed diets containing nonexcess levels of Leu.

Nε-(carboxymethyl)lysine-receptor for advanced glycation end product axis is a key modulator of obesity-induced dysregulation of adipokine expression and insulin resistance.

OBJECTIVE: Dysregulation of inflammatory adipokines by the adipose tissue plays an important role in obesity-associated insulin resistance. Pathways leading to this dysregulation remain largely unknown. We hypothesized that the receptor for advanced glycation end products (RAGE) and the ligand N(ε)-(carboxymethyl)lysine (CML) are increased in adipose tissue and, moreover, that activation of the CML-RAGE axis plays an important role in obesity-associated inflammation and insulin resistance. APPROACH AND RESULTS: In this study, we observed a strong CML accumulation and increased expression of RAGE in adipose tissue in obesity. We confirmed in cultured human preadipocytes that adipogenesis is associated with increased levels of CML and RAGE. Moreover, CML induced a dysregulation of inflammatory adipokines in adipocytes via a RAGE-dependent pathway. To test the role of RAGE in obesity-associated inflammation further, we constructed an obese mouse model that is deficient for RAGE (ie, RAGE(-/-)/Leptr(Db-/-) mice). RAGE(-/-)/Leptr(Db-/-) mice displayed an improved inflammatory profile and glucose homeostasis when compared with RAGE(+/+)/Leptr(Db-/-) mice. In addition, CML was trapped in adipose tissue in RAGE(+/+)/Leptr(Db-/-) mice but not in RAGE(-/-)/Leptr(Db-/-). RAGE-mediated trapping in adipose tissue provides a mechanism underlying CML accumulation in adipose tissue and explaining decreased CML plasma levels in obese subjects. Decreased CML plasma levels in obese individuals were strongly associated with insulin resistance. CONCLUSIONS: RAGE-mediated CML accumulation in adipose tissue and the activation of the CML-RAGE axis are important mechanisms involved in the dysregulation of adipokines in obesity, thereby contributing to the development of obesity-associated insulin resistance.

Potential role of pentosidine on susceptibility to small airway closure in elderly and smoking asthma.

BACKGROUND: Small airway closure in asthma is determined by a complex interaction of structural and functional characteristics including lung elastic recoil. Recently, we determined that loss of elastic recoil might be attributable to pentosidine level in the airways. This study was designed to investigate the influences of aging and smoking on small airway closure in asthma. METHODS: Sixty-one patients with asthma (20 non-smoking young adult, 23 non-smoking elderly, and 18 smoking young adult) and 36 control subjects (12 non-smoking young adult, 11 non-smoking elderly, and 13 smoking young adult) were included. We assessed airway responses during methacholine provocation and calculated the closing index. In addition, we measured pentosidine levels in induced sputum from all study subjects. RESULTS: Pentosidine levels in induced sputum were markedly higher in asthmatic patients than in controls. In control subjects, the intergroup differences in pentosidine level among 3 subgroups were significant. Similarly, pentosidine levels were significantly higher in non-smoking elderly and smoking young adult asthmatics than in non-smoking young adult asthmatics. There was no significant difference in pentosidine levels between non-smoking elderly and smoking young adult asthmatics. The closing index was also significantly higher in non-smoking elderly and smoking young adult asthmatics than in non-smoking young adult asthmatics. Moreover, pentosidine levels in non-smoking elderly and smoking young adult asthmatics were closely correlated with closing index. CONCLUSIONS: We determined the correlation of pentosidine level with susceptibility to small airway closure in elderly and smoking asthmatics. Our results might facilitate the understanding of elderly and smoking asthma.

Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a).

Apo(a), the distinguishing protein component of lipoprotein(a) [Lp(a)], exhibits sequence similarity to plasminogen and can inhibit binding of plasminogen to cell surfaces. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. The ability of apo(a) to inhibit pericellular plasminogen activation on vascular cells was therefore evaluated. Two isoforms of apo(a), 12K and 17K, were found to significantly decrease tissue-type plasminogen activator-mediated plasminogen activation on human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes and macrophages. Lp(a) purified from human plasma decreased plasminogen activation on THP-1 monocytes and HUVECs but not on THP-1 macrophages. Removal of kringle V or the strong lysine binding site in kringle IV10 completely abolished the inhibitory effect of apo(a). Treatment with carboxypeptidase B to assess the roles of carboxyl-terminal lysines in cellular receptors leads in most cases to decreases in plasminogen activation as well as plasminogen and apo(a) binding; however, inhibition of plasminogen activation by apo(a) was unaffected. Our findings directly demonstrate that apo(a) inhibits pericellular plasminogen activation in all three cell types, although binding of apo(a) to cell-surface receptors containing carboxyl-terminal lysines does not appear to play a major role in the inhibition mechanism.

Rumen-protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.

The objective of this experiment was to evaluate the effect of supplementing a metabolizable protein (MP)-deficient diet with rumen-protected (RP) Lys, Met, and specifically His on dairy cow performance. The experiment was conducted for 12 wk with 48 Holstein cows. Following a 2-wk covariate period, cows were blocked by DIM and milk yield and randomly assigned to 1 of 4 diets, based on corn silage and alfalfa haylage: control, MP-adequate diet (ADMP; MP balance: +9 g/d); MP-deficient diet (DMP; MP balance: -317 g/d); DMP supplemented with RPLys (AminoShure-L, Balchem Corp., New Hampton, NY) and RPMet (Mepron; Evonik Industries AG, Hanau, Germany; DMPLM); and DMPLM supplemented with an experimental RPHis preparation (DMPLMH). The analyzed crude protein content of the ADMP and DMP diets was 15.7 and 13.5 to 13.6%, respectively. The apparent total-tract digestibility of all measured nutrients, plasma urea-N, and urinary N excretion were decreased by the DMP diets compared with ADMP. Milk N secretion as a proportion of N intake was greater for the DMP diets compared with ADMP. Compared with ADMP, dry matter intake (DMI) tended to be lower for DMP, but was similar for DMPLM and DMPLMH (24.5, 23.0, 23.7, and 24.3 kg/d, respectively). Milk yield was decreased by DMP (35.2 kg/d), but was similar to ADMP (38.8 kg/d) for DMPLM and DMPLMH (36.9 and 38.5kg/d, respectively), paralleling the trend in DMI. The National Research Council 2001model underpredicted milk yield of the DMP cows by an average (±SE) of 10.3 ± 0.75 kg/d. Milk fat and true protein content did not differ among treatments, but milk protein yield was increased by DMPLM and DMPLMH compared with DMP and was not different from ADMP. Plasma essential amino acids (AA), Lys, and His were lower for DMP compared with ADMP. Supplementation of the DMP diets with RP AA increased plasma Lys, Met, and His. In conclusion, MP deficiency, approximately 15% below the National Research Council requirements from 2001, decreased DMI and milk yield in dairy cows. Supplementation of the MP-deficient diet with RPLys and RPMet diminished the difference in DMI and milk yield compared with ADMP and additional supplementation with RPHis eliminated it. As total-tract fiber digestibility was decreased with the DMP diets, but DMI tended to increase with RP AA supplementation, we propose that, similar to monogastric species, AA play a role in DMI regulation in dairy cows. Our data implicate His as a limiting AA in high-producing dairy cows fed corn silage- and alfalfa haylage-based diets, deficient in MP. The MP-deficient diets clearly increased milk N efficiency and decreased dramatically urinary N losses.

Translational control of cell division by Elongator.

Elongator is required for the synthesis of the mcm(5)s(2) modification found on tRNAs recognizing AA-ending codons. In order to obtain a global picture of the role of Elongator in translation, we used reverse protein arrays to screen the fission yeast proteome for translation defects. Unexpectedly, this revealed that Elongator inactivation mainly affected three specific functional groups including proteins implicated in cell division. The absence of Elongator results in a delay in mitosis onset and cytokinesis defects. We demonstrate that the kinase Cdr2, which is a central regulator of mitosis and cytokinesis, is under translational control by Elongator due to the Lysine codon usage bias of the cdr2 coding sequence. These findings uncover a mechanism by which the codon usage, coupled to tRNA modifications, fundamentally contributes to gene expression and cellular functions.

Estimation of the tryptophan requirement in piglets by meta-analysis.

There is no consensus concerning the Trp requirement for piglets expressed relative to Lys on a standardized ileal digestible basis (SID Trp : Lys). A meta-analysis was performed to estimate the SID Trp : Lys ratio that maximizes performance of weaned piglets between 7 and 25 kg of BW. A database comprising 130 experiments on the Trp requirement in piglets was established. The nutritional values of the diets were calculated from the composition of feed ingredients. Among all experiments, 37 experiments were selected to be used in the meta-analysis because they were designed to express the Trp requirement relative to Lys (e.g. Lys was the second-limiting amino acid in the diet) while testing at least three levels of Trp. The linear-plateau (LP), curvilinear-plateau (CLP) and asymptotic (ASY) models were tested to estimate the SID Trp : Lys requirement using average daily gain (ADG), average daily feed intake (ADFI) and gain-to-feed ratio (G : F) as response criteria. A multiplicative trial effect was included in the models on the plateau value, assuming that the experimental conditions affected only this parameter and not the requirement or the shape of the response to Trp. Model choice appeared to have an important impact on the estimated requirement. Using ADG and ADFI as response criteria, the SID Trp : Lys requirement was estimated at 17% with the LP model, at 22% with the CLP model and at 26% with the ASY model. Requirement estimates were slightly lower when G : F was used as response criterion. The Trp requirement was not affected by the composition of the diet (corn v. a mixture of cereals). The CLP model appeared to be the best-adapted model to describe the response curve of a population. This model predicted that increasing the SID Trp : Lys ratio from 17% to 22% resulted in an increase in ADG by 8%.

Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P4-ATPase ATP8A2.

ATP8A2 is a P(4)-ATPase ("flippase") located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P(4)-ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp(416)) in the P-type ATPase signature sequence and exists in E(1)P and E(2)P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp(196), Glu(198), Lys(873), and Asn(874) in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of E(2)P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K(+) activation of dephosphorylation in Na(+),K(+)-ATPase. Lys(873) mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys(873), located in transmembrane segment M5 at a "hot spot" for cation binding in Ca(2+)-ATPase and Na(+),K(+)-ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys(865) is unimportant for aminophospholipid sensitivity. Binding of Na(+), H(+), K(+), Cl(-), or Ca(2+) to the E(1) form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.

Many keys to push: diversifying the 'readership' of plant homeodomain fingers.

Covalent histone modifications-referred to as the 'histone code', are recognized by a wealth of effector or 'reader' modules, representing one of the most fundamental epigenetic regulatory mechanisms that govern the structure and function of our genome. Recent progresses on combinatorial readout of such 'histone code' promote us to reconsider epigenetic regulation as a more complicated theme than we originally anticipated. In particular, plant homeodomain (PHD) fingers, which are evolved with fine-tuned residue composition and integrated or paired with other reader modules, display remarkably diverse 'readership' other than its founding-member target, histone H3 trimethylation on lysine 4 (H3K4me3). In this review, we detail the latest progresses of PHD finger research, especially from the perspective of structural biology, and highlight the versatile binding features and biological significance of PHD fingers.

N(ɛ)-(carboxymethyl)lysine modification of elastin alters its biological properties: implications for the accumulation of abnormal elastic fibers in actinic elastosis.

Accumulation of degenerated elastic fibers in the sun-exposed skin designated as actinic elastosis is a histological hallmark of photodamaged skin. Previous studies have indicated that the elastic fibers of actinic elastosis interact with lysozyme and are modified by N(ɛ)-(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGEs). We studied here how CML modification of elastin is involved in the pathogenesis of actinic elastosis. The CML-modified insoluble elastin became resistant to neutrophil elastase digestion, which was reversed by treatment with aminoguanidine, a potent inhibitor of AGE formation. In a temperature-dependent aggregation assay, CML-modified elastin rapidly formed self-aggregates, the size of which was larger than unmodified elastin. The elastic fiber sheets prepared from CML-modified α-elastin showed 3D wider diameter, tortuous appearance, and decreased elasticity on tensile tests. The CML-modified α-elastin, but not unmodified α-elastin, was found to bind to lysozyme in vitro, supporting the immunohistochemical findings that the antibodies for lysozyme and CML reacted simultaneously with the elastic fibers of actinic elastosis and UV-irradiated skin. The glycated elastin is likely to cause the accumulation of abnormally aggregated elastic fibers and unusual interaction with lysozyme in actinic elastosis.

Role of uremic toxins and oxidative stress in the development of chronic kidney disease-mineral and bone disorder.

The kidney plays an important role in the regulation of mineral metabolism. As kidney function declines, there is a progressive deterioration in mineral homeostasis, along with various abnormalities, including bone disease and vascular calcification, which has recently been named as "Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD)." Although the precise mechanisms of this systemic disorder remain to be elucidated, accumulating evidence suggest that uremic toxins contribute substantially to the development of CKD-MBD, partly through evoking oxidative stress in the bone and cardiovascular systems. This brief review summarizes recent work on the role of uremic toxins and oxidative stress in the development of CKD-MBD.

Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA.

Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164)) is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS) polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1(K185)) was identified as the only topological equivalent of PCNA(K164). To investigate a potential role of posttranslational modifications of Rad1(K185) in DNA damage management, we here generated a mouse model with a conditional deletable Rad1(K185R) allele. The Rad1(K185) residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1(K185) is not a functional counterpart of PCNA(K164).

Cytosolic lysine residues enhance anterograde transport and activation of the erythropoietin receptor.

Lysine residues are key residues in many cellular processes, in part due to their ability to accept a wide variety of post-translational modifications. In the present study, we identify the EPO-R [EPO (erythropoietin) receptor] cytosolic lysine residues as enhancers of receptor function. EPO-R drives survival, proliferation and differentiation of erythroid progenitor cells via binding of its ligand EPO. We mutated the five EPO-R cytosolic lysine residues to arginine residues (5KR EPO-R), eliminating putative lysine-dependent modifications. Overexpressed 5KR EPO-R displayed impaired ubiquitination and improved stability compared with wt (wild-type) EPO-R. Unexpectedly, fusion proteins consisting of VSVGtsO45 (vesicular stomatitis virus glycoprotein temperature-sensitive folding mutant) with wt or 5KR EPO-R cytosolic domains demonstrated delayed glycan maturation kinetics upon substitution of the lysine residues. Moreover, VSVG-wt EPO-R, but not VSVG-5KR EPO-R, displayed endoplasmic reticulum-associated ubiquitination. Despite similar cell-surface EPO-binding levels of both receptors and the lack of EPO-induced ubiquitination by 5KR EPO-R, the lysine-less mutant produced weaker receptor activation and signalling than the wt receptor. We thus propose that EPO-R cytosolic lysine residues enhance receptor function, most probably through ubiquitination and/or other post-translational modifications.

L-leucine, L-methionine, and L-lysine are involved in the regulation of intermediary metabolism-related gene expression in rainbow trout hepatocytes.

Using rainbow trout hepatocytes stimulated with l-leucine, l-methionine, or l-lysine in the presence or absence of bovine insulin, we investigated the ability of these amino acids to mimic the effects of a pool of amino acids on protein kinase B (Akt)/target of rapamycin (TOR) signaling pathways and expression of 6 genes known to be subjected to insulin and/or amino acid regulation [glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), glucokinase (GK), pyruvate kinase (PK), fatty acid synthase (FAS), and serine dehydratase (SDH)]. Emphasis was placed on leucine, known to be a signaling molecule in mammals, and methionine and lysine that are essential amino acids limiting in plant-based diets for fish. In the presence of insulin, leucine (but not methionine or lysine) phosphorylated Akt and ribosomal protein S6 as previously observed with a pool of amino acids, suggesting that leucine might participate in the activation of the TOR pathway by amino acids in fish, as in mammals. G6Pase, PEPCK, GK, and SDH gene expression were higher in leucine-treated cells compared with control cells. Leucine combined with insulin reduced G6Pase gene expression by 90% and increased FAS gene expression > 4-fold compared with the control treatment. Methionine weakly decreased G6Pase, GK, and SDH gene expression and lysine weakly but significantly decreased the mRNA level of PEPCK. Thus, leucine regulated gluconeogenesis and lipogenesis, but not glycolysis, in the same way as a pool of amino acids. Methionine appeared to be involved in the regulation of specific genes, whereas lysine only had limited effects. These findings are particularly relevant regarding the involvement of amino acids in the regulation of metabolism-related gene expression.