Amino Acids (v.48, #5)

New concepts of microbial treatment processes for the nitrogen removal: effect of protein and amino acids degradation by Alejandro González-Martínez; Kadiya Calderón; Jesús González-López (1123-1130).
High concentrations of proteins and amino acids can be found in wastewater and wastewater stream produced in anaerobic digesters, having shown that amino acids could persist over different managements for nitrogen removal affecting the nitrogen removal processes. Nitrogen removal is completely necessary because of their implications and the significant adverse environmental impact of ammonium such as eutrophication and toxicity to aquatic life on the receiving bodies. In the last decade, the treatment of effluents with high ammonium concentration through anammox-based bioprocesses has been enhanced because these biotechnologies are cheaper and more environmentally friendly than conventional technologies. However, it has been shown that the presence of important amounts of proteins and amino acids in the effluents seriously affects the microbial autotrophic consortia leading to important losses in terms of ammonium oxidation efficiency. Particularly the presence of sulfur amino acids such as methionine and cysteine has been reported to drastically decrease the autotrophic denitrification processes as well as affect the microbial community structure promoting the decline of ammonium oxidizing bacteria in favor of other phylotypes. In this context we discuss that new biotechnological processes that improve the degradation of protein and amino acids must be considered as a priority to increase the performance of the autotrophic denitrification biotechnologies.
Keywords: Amino acids; Proteins; Nitrogen removal; Wastewater; Microbial population

Physiological and therapeutic effects of carnosine on cardiometabolic risk and disease by Estifanos Baye; Barbara Ukropcova; Jozef Ukropec; Alan Hipkiss; Giancarlo Aldini; Barbora de Courten (1131-1149).
Obesity, type 2 diabetes (T2DM) and cardiovascular disease (CVD) are the most common preventable causes of morbidity and mortality worldwide. They represent major public health threat to our society. Increasing prevalence of obesity and T2DM contributes to escalating morbidity and mortality from CVD and stroke. Carnosine (β-alanyl-l-histidine) is a dipeptide with anti-inflammatory, antioxidant, anti-glycation, anti-ischaemic and chelating roles and is available as an over-the-counter food supplement. Animal evidence suggests that carnosine may offer many promising therapeutic benefits for multiple chronic diseases due to these properties. Carnosine, traditionally used in exercise physiology to increase exercise performance, has potential preventative and therapeutic benefits in obesity, insulin resistance, T2DM and diabetic microvascular and macrovascular complications (CVD and stroke) as well as number of neurological and mental health conditions. However, relatively little evidence is available in humans. Thus, future studies should focus on well-designed clinical trials to confirm or refute a potential role of carnosine in the prevention and treatment of chronic diseases in humans, in addition to advancing knowledge from the basic science and animal studies.
Keywords: Carnosine; Skeletal muscle; Obesity; Insulin resistance; Type 2 diabetes; Energy expenditure; Atherosclerosis; Cholesterol; Ischaemia

Protein/peptide hormones are the largest group of endogenous signaling molecules and exert various biological functions by binding to specific cell membrane receptors. To study the interactions between these hormones and their receptors, quantitative ligand–receptor binding assays have been widely used for decades. However, the assays conventionally relied on the use of radioligands, which have some major drawbacks and can only be used in laboratories with a radioactive material license. We recently developed novel bioluminescent binding assays for several protein/peptide hormones using the brightest bioluminescent reporter known to date, nanoluciferase (NanoLuc). The NanoLuc reporter can be either chemically conjugated to an appropriate position, or genetically fused at one terminus, of protein/peptide hormones. Compared to conventional radioligands, these bioluminescent ligands have higher sensitivity, better safety, and longer shelf lives, and thus, represent a novel class of non-radioactive tracers for quantitative receptor binding assays. In the present review, we provide some general considerations and specific examples for setting up the bioluminescent binding assays. Such techniques can be applied to other protein/peptide hormones in future to facilitate their interaction studies with their receptors.
Keywords: Protein; Peptide; Ligand; Receptor; Binding; Bioluminescence; NanoLuc

Amino acids in the cultivation of mammalian cells by Andrew Salazar; Michael Keusgen; Jörg von Hagen (1161-1171).
Amino acids are crucial for the cultivation of mammalian cells. This importance of amino acids was realized soon after the development of the first cell lines, and a solution of a mixture of amino acids has been supplied to cultured cells ever since. The importance of amino acids is further pronounced in chemically defined mammalian cell culture media, making the consideration of their biological and chemical properties necessary. Amino acids concentrations have been traditionally adjusted to their cellular consumption rates. However, since changes in the metabolic equilibrium of amino acids can be caused by changes in extracellular concentrations, metabolomics in conjunction with flux balance analysis is being used in the development of culture media. The study of amino acid transporters is also gaining importance since they control the intracellular concentrations of these molecules and are influenced by conditions in cell culture media. A better understanding of the solubility, stability, dissolution kinetics, and interactions of these molecules is needed for an exploitation of these properties in the development of dry powdered chemically defined media for mammalian cells. Due to the complexity of these mixtures however, this has proven to be challenging. Studying amino acids in mammalian cell culture media will help provide a better understanding of how mammalian cells in culture interact with their environment. It would also provide insight into the chemical behavior of these molecules in solutions of complex mixtures, which is important in the understanding of the contribution of individual amino acids to protein structure.
Keywords: Amino acid; Bioprocess; Biotechnology; Cell culture media; Media formulation

Proteome profiling reveals tissue-specific protein expression in male and female accessory glands of the silkworm, Bombyx mori by Zhaoming Dong; Xiaohuan Wang; Yan Zhang; Liping Zhang; Quanmei Chen; Xiaolu Zhang; Ping Zhao; Qingyou Xia (1173-1183).
Male accessory gland (MAG) and female accessory gland (FAG) of the reproductive system are, respectively, responsible for producing seminal proteins and adhesive proteins during copulation and ovulation. Seminal proteins are ejaculated to female along with sperms, whereas adhesive proteins are excreted along with eggs. Proteins from the male and female reproductive organs are usually indicative of rapid adaptive evolution. Understanding the reproductive isolation and species divergence requires identifying reproduction-related proteins from many different species. Here, we present our proteomic analyses of male and female accessory glands of the silkworm, Bombyx mori. Using LC/MS–MS, we identified 2133 MAG proteins and 1872 FAG proteins. In total, 652 proteins were significant more abundant in the MAG than in the FAG, including growth factors, odorant-binding proteins, enzymes, and proteins of unknown function. Growth factors and odorant-binding proteins are potential signaling molecules, whereas most of proteins of unknown function were found to be Lepidoptera-specific proteins with high evolutionary rates. Microarray experiments and semi-quantitative RT-PCR validated that MAG-specific proteins were expressed exclusively in male moths. Totally, 192 proteins were considered as FAG-specific proteins, including protease inhibitors, enzymes, and other proteins. Protease inhibitors were found to be the most abundant FAG-specific proteins, which may protect eggs from infection by inhibiting pathogen-derived proteases. These results provide comprehensive insights into copulation and oviposition. Moreover, the newly identified Lepidoptera-specific MAG proteins provide useful data for future research on the evolution of reproductive proteins in insects.
Keywords: Bombyx mori ; Reproduction; Accessory gland; Protease inhibitor; Growth factor; Odorant-binding protein

CK1δ kinase activity is modulated by protein kinase C α (PKCα)-mediated site-specific phosphorylation by Zhigang Meng; Joachim Bischof; Chiara Ianes; Doris Henne-Bruns; Pengfei Xu; Uwe Knippschild (1185-1197).
Cellular signal transduction components are usually regulated not only on transcriptional or translational level, but also by posttranslational modifications. Among these, reversible phosphorylation represents the most abundant modification. In general, phosphorylation events are essential for regulating the activity of central signal transduction proteins, also including kinases itself. Members of the CK1 family can be found as central signal transduction proteins in numerous cellular pathways. Due to its wide variety of cellular functions the activity of CK1 family members has to be tightly regulated. We previously reported that PKA and Chk1 are able to phosphorylate CK1δ within its C-terminal regulatory domain, consequently resulting in altered CK1 kinase activity. In the present study, we show by several methods that protein kinase C α (PKCα) as well is able to phosphorylate CK1δ at its C-terminally located residues S328, T329, and S370. Furthermore, we analyze the functional consequences of PKCα-mediated phosphorylation on CK1δ kinase activity. Mutation of S328, T329, or S370 to alanine dramatically alters the kinetic parameters of CK1δ. By using the PKCα-specific inhibitor Go-6983 in a selected cell culture model, we finally show that the in vitro detected regulatory connection between PKCα and CK1δ is also relevant in the cellular context. Taken together, these data contribute to a deeper understanding of cellular signal transduction networks thereby helping to form a basis for the development of future therapeutic concepts.
Keywords: Casein kinase 1 (CK1); Protein kinase C (PKC); Phosphorylation; Inhibitor; Mass spectrometry (MS)

Taurine content in different brain structures during ageing: effect on hippocampal synaptic plasticity by Luz M. Suárez; María-Dolores Muñoz; Rafael Martín del Río; José M. Solís (1199-1208).
A reduction in taurine content accompanies the ageing process in many tissues. In fact, the decline of brain taurine levels has been associated with cognitive deficits whereas chronic administration of taurine seems to ameliorate age-related deficits such as memory acquisition and retention. In the present study, using rats of three age groups (young, adult and aged) we determined whether the content of taurine and other amino acids (glutamate, serine, glutamine, glycine, alanine and GABA) was altered during ageing in different brain areas (cerebellum, cortex and hippocampus) as well non-brain tissues (heart, kidney, liver and plasma). Moreover, using hippocampal slices we tested whether ageing affects synaptic function and plasticity. These parameters were also determined in aged rats fed with either taurine-devoid or taurine-supplemented diets. With age, we found heterogeneous changes in amino acid content depending on the amino acid type and the tissue. In the case of taurine, its content was reduced in the cerebellum of adult and aged rats, but it remained unchanged in the hippocampus, cortex, heart and liver. The synaptic response amplitude decreased in aged rats, although the late phase of long-term synaptic potentiation (late-LTP), a taurine-dependent process, was not altered. Our study highlights the stability of taurine content in the hippocampus during ageing regardless of whether taurine was present in the diet, which is consistent with the lack of changes detected in late-LTP. These results indicate that the beneficial effects of taurine supplementation might be independent of the replenishment of taurine stores.
Keywords: Ageing; Nutrition; Synaptic plasticity; LTP; d-Serine; Brain

Characterization of aromatic aminotransferases from Ephedra sinica Stapf by Korey Kilpatrick; Agnieszka Pajak; Jillian M. Hagel; Mark W. Sumarah; Efraim Lewinsohn; Peter J. Facchini; Frédéric Marsolais (1209-1220).
Ephedra sinica Stapf (Ephedraceae) is a broom-like shrub cultivated in arid regions of China, Korea and Japan. This plant accumulates large amounts of the ephedrine alkaloids in its aerial tissues. These analogs of amphetamine mimic the actions of adrenaline and stimulate the sympathetic nervous system. While much is known about their pharmacological properties, the mechanisms by which they are synthesized remain largely unknown. A functional genomics platform was established to investigate their biosynthesis. Candidate enzymes were obtained from an expressed sequence tag collection based on similarity to characterized enzymes with similar functions. Two aromatic aminotransferases, EsAroAT1 and EsAroAT2, were characterized. The results of quantitative reverse transcription-polymerase chain reaction indicated that both genes are expressed in young stem tissue, where ephedrine alkaloids are synthesized, and in mature stem tissue. Nickel affinity-purified recombinant EsAroAT1 exhibited higher catalytic activity and was more homogeneous than EsAroAT2 as determined by size-exclusion chromatography. EsAroAT1 was highly active as a tyrosine aminotransferase with α-ketoglutarate followed by α-ketomethylthiobutyrate and very low activity with phenylpyruvate. In the reverse direction, catalytic efficiency was similar for the formation of all three aromatic amino acids using l-glutamate. Neither enzyme accepted putative intermediates in the ephedrine alkaloid biosynthetic pathway, S-phenylacetylcarbinol or 1-phenylpropane-1,2-dione, as substrates.
Keywords: Ephedra sinensis Stapf Ephedraceae; Aminotransferase; Aromatic amino acids; l-Tyrosine; Pyridoxal 5′-phosphate; Transcriptome analysis

Regulation of tyrosine hydroxylase is preserved across different homo- and heterodimeric 14-3-3 proteins by Sadaf Ghorbani; Agnete Fossbakk; Ana Jorge-Finnigan; Marte I. Flydal; Jan Haavik; Rune Kleppe (1221-1229).
Tyrosine hydroxylase (TH) is regulated by members of the 14-3-3 protein family. However, knowledge about the variation between 14-3-3 proteins in their regulation of TH is still limited. We examined the binding, effects on activation and dephosphorylation kinetics of tyrosine hydroxylase (TH) by abundant midbrain 14-3-3 proteins (β, η, ζ, γ and ε) of different dimer composition. All 14-3-3 homodimers and their respective 14-3-3ε-heterodimers bound with similar high affinity (K d values of 1.4–3.8 nM) to serine19 phosphorylated human TH (TH-pS19). We similarly observed a consistent activation of bovine (3.3- to 4.4-fold) and human TH-pS19 (1.3–1.6 fold) across all the different 14-3-3 dimer species, with homodimeric 14-3-3γ being the strongest activator. Both hetero- and homodimers of 14-3-3 strongly inhibited dephosphorylation of TH-pS19, and we speculate if this is an important homeostatic mechanism of 14-3-3 target-protein regulation in vivo. We conclude that TH is a robust interaction partner of different 14-3-3 dimer types with moderate variability between the 14-3-3 dimers on their regulation of TH.
Keywords: 14-3-3; Heterodimer; Isoform; Tyrosine hydroxylase; Activation

New studies about the insertion mechanism of Thymosin α1 in negative regions of model membranes as starting point of the bioactivity by Walter Mandaliti; Ridvan Nepravishta; Paola Sinibaldi Vallebona; Francesca Pica; Enrico Garaci; Maurizio Paci (1231-1239).
Thymosin α1 is a peptidic hormone already used in the therapy of several diseases. Until now, the description of the precise receptor and mechanism for its action still remains elusive. The interaction of Thymosin α1, which is unstructured in water solution, has been recently studied in sodium dodecylsulphate micellar systems and it was reported that Thymosin α1 inserts in micelle assuming a conformation with two tracts of helix with a structural break in between. An investigation of its interaction both with micelles of dodecylphosphocholine alone and with mixed dodecylphosphocholine-sodium dodecylsulphate micelles is here reported. In these environments the results indicate that Thymosin α1 in phospholipidic membrane exposing choline polar heads interacts by aspecific modality and, oppositely, in the mixed dodecylphosphocholine-sodium dodecylsulphate micelles an insertion in the micellar hydrophobic region conformationally similar to that found in sodium dodecylsulphate micelles occurs. In presence of mixed micelles the insertion and structuration occur in preferred regions when the membrane models are negatively charged. From the point of view of the mechanism of action, insertion its N terminus in negative regions of membrane led to hypothesize that this process would be similar to a binding to phosphatidylserine exposed like in apoptotic cells. Thymosin α1 when inserted may interact with nearby proteins and/or receptors acting as effector and causing a biological signaling cascade. The recent attention to the phosphatidylserine exposure in cells may enforce the interest for these findings.
Keywords: Thymosin α1; Thymic hormons; NMR spectroscopy; Model membranes; Peptide

The stereochemical effect of SMAP-29 and SMAP-18 on bacterial selectivity, membrane interaction and anti-inflammatory activity by Binu Jacob; Ganesan Rajasekaran; Eun Young Kim; Il-Seon Park; Jeong-Kyu Bang; Song Yub Shin (1241-1251).
Sheep myeloid antimicrobial peptide-29 (SMAP-29) is a cathelicidin-related antimicrobial peptide derived from sheep myeloid cells. In order to investigate the effects of l-to-d-amino acid substitution in SMAP-29 on bacterial selectivity, membrane interaction and anti-inflammatory activity, we synthesized its two d-enantiomeric peptides (SMAP-29-E1 and SMAP-29-E2 containing d-Ile and d-allo-Ile, respectively) and two diastereomeric peptides (SMAP-29-D1 and SMAP-29-D2). Additionally, in order to address the effect of l-to-d-amino acid substitution in the N-terminal helical peptide of SMAP-29 (named SMAP-18) on antimicrobial activity, we synthesized its two d-enantiomeric peptides (SMAP-18-E1 and SMAP-18-E2), which are composed of d-amino acids entirely. l-to-d-amino acid substitution in membrane-targeting AMP, SMAP-29 did not affect its antimicrobial activity. However, d-allo-Ile containing-SMAP-29-E2 and SMAP-29-D2 exhibited less hemolytic activity compared to d-Ile containing-SMAP-29-E1 and SMAP-29-D1, respectively. l-to-d-amino acid substitution in intracellular targeting-AMPs, SMAP-18 and buforin-2 improved antimicrobial activity by 2- to eightfold. The improved antimicrobial activity of the d-isomers of SMAP-18 and buforin-2 seems to be due to the stability against proteases inside bacterial cells. Membrane depolarization and dye leakage suggested that the membrane-disruptive mode of SMAP-29-D1 and SMAP-29-D2 is different from that of SMAP-29, SMAP-29-E1, and SMAP-29-E2. l-to-d-amino acid substitution in SMAP-29 improved anti-inflammatory activity in LPS-stimulated RAW 264.7 cells. In summary, we propose here that d-allo-Ile substitution is a more powerful strategy for increasing bacterial selectivity than d-Ile substitution in the design of d-enantiomeric and diastereomeric AMPs. SMAP-29-D1, and SMAP-29-D2 with improved bacterial selectivity and anti-inflammatory activity can serve as promising candidates for the development of anti-inflammatory and antimicrobial agents.
Keywords: SMAP-29/SMAP-18; l-to-d-amino acid substitution; Bacterial selectivity; Anti-inflammatory activity; Protease stability

Aspergillus niger PA2: a novel strain for extracellular biotransformation of l-tyrosine into l-DOPA by Pragati Agarwal; Nidhi Pareek; Swati Dubey; Jyoti Singh; R. P. Singh (1253-1262).
l-DOPA (3,4-dihydroxyphenyl-l-alanine), an amino acid derivative is the most widely used drug of choice for the treatment of Parkinson’s disease and other neurologic injuries. The present study deals with the elevated biochemical transformation of l-tyrosine to l-DOPA by Aspergillus niger PA2, a potent tyrosinase producer, isolated from decomposed food wastes. This appears to be the first report on A. niger as a notable extracellular tyrosinase producer. The extracellular tyrosinase activity produced remarkably higher levels of l-DOPA, i.e. 2.44 mg mL−1 when the media was supplemented with 5 mg mL−1 l-tyrosine. The optimum pH for tyrosinase production was 6.0, with the maximal l-DOPA production at the same pH. The product thus produced was analyzed by thin-layer chromatography, UV spectroscopy, high-performance liquid chromatography and Fourier transform infrared spectroscopy, that had denoted this to be l-DOPA. Kinetic parameters viz. Y p/s, Q s and Q p had further indicated the notable levels of production. Thus, Aspergillus niger PA2 could be a promising resource and may be further exploited for large-scale production of l-DOPA.
Keywords: Tyrosinase; l-DOPA; Biotransformation; Aspergillus niger

Hepatocyte apoptosis plays a key role in the pathogenesis of immune-mediated hepatitis. However, the detailed mechanisms of apoptosis signaling are still unclear and effective therapeutic drugs for hepatitis have been explored. Here, we show that tryptophan (Trp) suppressed IFN-γ-mediated hepatic apoptosis in vitro. Trp inhibited the downstream apoptotic events of mitochondria disruption, such as cell death and caspase-3 activation, while it did not influence upstream signaling including STAT1 activation and IRF1 expression. Trp suppressed reactive oxygen species (ROS) generation at the mitochondria. IFN-γ induced ROS in mitochondria by inhibiting complex I and III, but not II. This ROS generation by IFN-γ required de novo protein synthesis. Trp showed relatively weak direct scavenging activity but antagonized IFN-γ against the suppression of complex I. In addition, Trp increased the expression of the Nrf2-dependent antioxidant genes NQO1, HO-1 and GCS in hepatocytes both in vitro and in vivo. Finally, the administration of Trp in an acetaminophen-induced ROS-dependent hepatitis model suppressed the liver injury in vivo. Thus, Trp protects hepatocytes from ROS-dependent cell injury via multiple pathways. This study suggests Trp as a therapeutic antioxidant drug for hepatitis and a regulator for Nrf2-dependent genes.
Keywords: Tryptophan; Hepatocyte; Reactive oxygen species; Apoptosis; Nrf2

The interaction of central nitrergic and GABAergic systems on food intake in neonatal layer-type chicks by Kasra Mokhtarpouriani; Morteza Zendehdel; Hossein Jonaidi; Vahab Babapour; Parviz Shayan (1275-1283).
Most physiological behaviors such as food intake are controlled by the hypothalamus and its nuclei. It has been demonstrated that injection of the paraventricular nucleus of the hypothalamus with nitric oxide (NO) donors elicited changes in the concentration of some amino acids, including GABA. Also, central nitrergic and GABAergic systems are known to provide inputs to the paraventricular nucleus and are involved in food intake control. Therefore, the present study examines the probable interaction of central nitrergic and GABAergic systems on food intake in neonatal layer-type chicks. The results of this study showed that intracerebroventricular (ICV) injection of l-arginine (400 and 800 nmol), as a NO donor, significantly decreased food intake (P < 0.001), but ICV injection of Nω-Nitro-l-arginine methyl ester (L-NAME) (200 and 400 nmol), a NO synthesis inhibitor, increased food intake (P < 0.001). In addition, the orexigenic effect of gaboxadol (0.2 µg), a GABAA agonist, was significantly attenuated in ICV co-injection of l-arginine (200 nmol) and gaboxadol (0.2 µg) (P < 0.001), but it was significantly amplified in ICV co-injection of L-NAME (100 nmol) and gaboxadol (0.2 µg) (P < 0.001). On the other hand, the orexigenic effect of baclofen (0.2 µg), a GABAB agonist, did not change in ICV co-injection of l-arginine (200 nmol) or L-NAME (100 nmol) with baclofen (0.2 µg) (P > 0.05). Also, the hypophagic effect of l-arginine (800 nmol) was significantly amplified in ICV co-injection of picrotoxin (0.5 µg), a GABAA antagonist, or CGP54626 (21 ng), a GABAB antagonist, with l-arginine (800 nmol) (P < 0.001). These results probably suggest an interaction of central nitrergic and GABAergic systems on food intake in neonatal layer-type chicks and GABAA receptors play a major role in this interaction.
Keywords: NO; GABA; Food intake; Neonatal layer-type chick

Nitrogen fixation of the nodule of soybean is highly sensitive to oxygen deficiency such as provoked by waterlogging of the root system. This study aimed to evaluate the effects of flooding on N metabolism in nodules of soybean. Flooding resulted in a marked decrease of asparagine (the most abundant amino acid) and a concomitant accumulation of γ-aminobutyric acid (GABA). Flooding also resulted in a strong reduction of the incorporation of 15N2 in amino acids. Nodule amino acids labelled before flooding rapidly lost 15N during flooding, except for GABA, which initially increased and declined slowly thereafter. Both nitrogenase activity and the expression of nifH and nifD genes were strongly decreased on flooding. Expression of the asparagine synthetase genes SAS1 and SAS2 was reduced, especially the former. Expression of genes encoding the enzyme glutamic acid decarboxylase (GAD1, GAD4, GAD5) was also strongly suppressed except for GAD2 which increased. Almost all changes observed during flooding were reversible after draining. Possible changes in asparagine and GABA metabolism that may explain the marked fluctuations of these amino acids during flooding are discussed. It is suggested that the accumulation of GABA has a storage role during flooding stress.
Keywords: Amino acids; Nitrogenase; 15N2 incorporation; NifH ; Asparagine; GABA

Quantitative proteomics analysis reveals glutamine deprivation activates fatty acid β-oxidation pathway in HepG2 cells by Baisheng Long; Rodiallah Muhamad; Guokai Yan; Jie Yu; Qiwen Fan; Zhichang Wang; Xiuzhi Li; Agung Purnomoadi; Joelal Achmadi; Xianghua Yan (1297-1307).
Glutamine, a multifunctional amino acid, functions in nutrient metabolism, energy balance, apoptosis, and cell proliferation. Lipid is an important nutrient and controls a broad range of physiological processes. Previous studies have demonstrated that glutamine can affect lipolysis and lipogenesis, but the effect of glutamine on the detailed lipid metabolism remains incompletely understood. Here, we applied the quantitative proteomics approach to estimate the relative abundance of proteins in HepG2 cells treated by glutamine deprivation. The results showed that there were 212 differentially abundant proteins in response to glutamine deprivation, including 150 significantly increased proteins and 62 significantly decreased proteins. Interestingly, functional classification showed that 43 differentially abundant proteins were related to lipid metabolism. Further bioinformatics analysis and western blotting validation revealed that lipid accumulation may be affected by β-oxidation of fatty acid induced by glutamine deprivation in HepG2 cells. Together, our results may provide the potential for regulating lipid metabolism by glutamine in animal production and human nutrition. The MS data have been deposited to the ProteomeXchange Consortium with identifier PXD003387.
Keywords: Glutamine; Lipid metabolism; Fatty acid; Quantitative proteomics; HepG2 cells

Incorporation of tryptophan analogues into the lantibiotic nisin by Liang Zhou; Jinfeng Shao; Qian Li; Auke J. van Heel; Marcel P. de Vries; Jaap Broos; Oscar P. Kuipers (1309-1318).
Lantibiotics are posttranslationally modified peptides with efficient inhibitory activity against various Gram-positive bacteria. In addition to the original modifications, incorporation of non-canonical amino acids can render new properties and functions to lantibiotics. Nisin is the most studied lantibiotic and contains no tryptophan residues. In this study, a system was constructed to incorporate tryptophan analogues into nisin, which included the modification machinery (NisBTC) and the overexpression of tryptophanyl-tRNA synthetase (TrpRS). Tryptophan and three different tryptophan analogues (5-fluoroTrp (5FW), 5-hydroxyTrp (5HW) and 5-methylTrp (5MeW)) were successfully incorporated at four different positions of nisin (I1W, I4W, M17W and V32W). The incorporation efficiency of tryptophan analogues into mutants I1W, M17W and V32W was over 97 %, while the mutant I4W showed relatively low incorporation efficiency (69–93 %). The variants with 5FW showed relatively higher production yield, while 5MeW-containing variants showed the lowest yield. The dehydration efficiency of serines or threonines was affected by the tryptophan mutants of I4W and V32W. The affinity of the peptides for the cation-ion exchange and reverse phase chromatography columns was significantly reduced when 5HW was incorporated. The antimicrobial activity of IIW and its 5FW analogue both decreased two times compared to that of nisin, while that of its 5HW analogue decreased four times. The 5FW analogue of I4W also showed two times decreased activity than nisin. However, the mutant M17W and its 5HW analogue both showed 32 times reduced activity relative to that of nisin.
Keywords: Lantibiotics; Nisin; Tryptophan analogues; Biosynthetic incorporation; Non-canonical amino acids

Generally, small peptides by themselves are weak to induce antibody responses. Toll-like receptor (TLR) ligands are attractive candidates of vaccine adjuvants to improve their antigenicity. The covalent conjugation of TLR ligands with antigens to produce self-adjuvanting peptide vaccine is a promising approach. Based on the structure of TLR7/8 ligands, a series of synthetic amino acids 6-imidazoquinolyl-norleucines were synthesized, wherein an imidazoquinoline structure as the TLR7/8 agonistic pharmacophores was constructed on the ε-NH2 group of Lys. Of them, 6-(4-amino-2-butyl-imidazoquinolyl)-norleucine showed the most potent TLR7 and TLR8 agonistic activities with EC50 values of 8.55 and 106 μM, respectively. Subsequently, mice were immunized with the influenza A virus M2e antigen mixed with or covalently conjugated to the TLR7/8 agonist amino acid, which led to induction of M2e specific antibody productions in the absence of other adjuvant. We successfully developed a novel efficient tool for self-adjuvanting peptide vaccines targeting TLR7/8.
Keywords: Adjuvant; Amino acid; Imidazoquinoline; Self-adjuvanting peptide vaccine; Toll-like receptor

Proteome-derived peptide library for the elucidation of the cleavage specificity of HF3, a snake venom metalloproteinase by Luciana Bertholim; André Zelanis; Ana K. Oliveira; Solange M. T. Serrano (1331-1335).
The Proteomic Identification of Cleavage Sites (PICS) approach was employed for profiling the substrate specificity of HF3, a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops jararaca. A tryptic peptide library from human plasma was subject to HF3 cleavage and amino acid occurrence for P6 to P6′ sites was mapped. 71 cleavage sites were detected and revealed a clear preference for leucine at P1′ position, followed by hydrophobic residues in P2′. PICS confirmed existing data on prime site specificity of SVMPs.
Keywords: Snake venom; Metalloproteinase; Proteomic identification of protease cleavage sites; Peptide bond specificity; Proteome derived peptide library; HF3

Erratum to: Co-regulation of mitochondrial respiration by proline dehydrogenase/oxidase and succinate by Chad N. Hancock; Wei Liu; W. Gregory Alvord; James M. Phang (1337-1338).