Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.     

Copper stress‐induced changes in leaf soluble proteome of Cu‐sensitive and tolerant Agrostis capillaris L. populations

Changes in leaf soluble proteome were explored in 3‐month‐old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1–50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO₄ spiked‐nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2‐DE (linear 4–7 pH gradient). Analysis of CCB‐stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC‐MS/MS. In both populations, Cu excess impacted both light‐dependent (OEE, cytochrome b6‐f complex, and chlorophyll a‐b binding protein), and ‐independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin‐NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S‐containing amino‐acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 ( http//proteomecentral.proteomexchange.org/dataset/PXD001930 ).     

Absolute protein quantification allows differentiation of cell‐specific metabolic routes and functions

Total protein approach (TPA) is a proteomic method that allows calculation of concentrations of individual proteins and groups of functionally related proteins in any protein mixture without spike‐in standards. Using the two‐step digestion–filter‐aided sample preparation method and LC‐MS/MS analysis, we generated comprehensive quantitative datasets of mouse intestinal mucosa, liver, red muscle fibers, brain, and of human plasma, erythrocytes, and tumor cells lines. We show that the TPA‐based quantitative data reflect well‐defined and specific physiological functions of different organs and cells, for example nutrient absorption and transport in intestine, amino acid catabolism and bile secretion in liver, and contraction of muscle fibers. Focusing on key metabolic processes, we compared metabolic capacities in different tissues and cells. In addition, we demonstrate quantitative differences in the mitochondrial proteomes. Providing insight into the abundances of mitochondrial metabolite transporters, we demonstrate that their titers are well tuned to cell‐specific metabolic requirements. This study provides for the first time a comprehensive overview of the protein hardware mediating metabolism in different mammalian organs and cells. The presented approach can be applied to any other system to study biological processes. All MS data have been deposited in the ProteomeXchange with identifier PXD001352 ( http://proteomecentral.proteomexchange.org/dataset/PXD001352 ).     

IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif

Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA. OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB. This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif. OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro. The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper. We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction. Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function. Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats. Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB[1-149], that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex". Preincubation of OrfAB[1-149] with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA. Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats.     

Methionine in proteins: The Cinderella of the proteinogenic amino acids

Methionine in proteins, apart from its role in the initiation of translation, is assumed to play a simple structural role in the hydrophobic core, in a similar way to other hydrophobic amino acids such as leucine, isoleucine, and valine. However, research from a number of laboratories supports the concept that methionine serves as an important cellular antioxidant, stabilizes the structure of proteins, participates in the sequence‐independent recognition of protein surfaces, and can act as a regulatory switch through reversible oxidation and reduction. Despite all these evidences, the role of methionine in protein structure and function is largely overlooked by most biochemists. Thus, the main aim of the current article is not so much to carry out an exhaustive review of the many and diverse processes in which methionine residues are involved, but to review some illustrative examples that may help the nonspecialized reader to form a richer and more precise insight regarding the role‐played by methionine residues in such processes.     

Phosphoproteomics of the goat milk fat globule membrane: New insights into lipid droplet secretion from the mammary epithelial cell

Mechanisms of milk lipid secretion are highly controversial. Analyzing the fine protein composition of the “milk fat globule membrane” (MFGM), the triple‐layered membrane surrounding milk lipid droplets (LDs) can provide mechanistic clues to better understand LD biosynthesis and secretion pathways in mammary epithelial cells (MECs). We therefore combined a high‐sensitive Q‐Exactive LC‐MS/MS analysis of MFGM‐derived peptides to the use of an in‐house database intended to improve protein identification in the goat species. Using this approach, we performed the identification of 442 functional groups of proteins in the MFGM from goat milk. To get a more dynamic view of intracellular mechanisms driving LD dynamics in the MECs, we decided to investigate for the first time whether MFGM proteins were phosphorylated. MFGM proteins were sequentially digested by lysine‐C and trypsin proteases and the resulting peptides were fractionated by a strong cation exchange chromatography. Titanium beads were used to enrich phosphopeptides from strong cation exchange chromatography eluted fractions. This approach lets us pinpoint 271 sites of phosphorylation on 124 unique goat MFGM proteins. Enriched GO terms associated with phosphorylated MFGM proteins were protein transport and actin cytoskeleton organization. Gained data are discussed with regard to lipid secretory mechanisms in the MECs. All MS data have been deposited in the ProteomeXchange with identifier PXD001039 ( http://proteomecentral.proteomexchange.org/dataset/PXD001039 ).     

BasT, a membrane-bound transducer protein for amino acid detection in Halobacterium salinarum

Halophilic archaea, such as eubacteria, use methyl-accepting chemotaxis proteins (MCPs) to sense their environment. We show here that BasT is a halobacterial transducer protein (Htp) responsible for chemotaxis towards five attractant amino acids. The C-terminus of the protein exhibits the highly conserved regions that are diagnostic for MCPs: the signalling domain for communication with the histidine kinase and the methylation sites that interact with the methylation/demethylation enzymes for adaptation. Hydropathy analysis predicts an enterobacterial-type transducer protein topology for BasT, with an extracellular putative ligand-binding domain flanked by two transmembrane helices and a cytoplasmic domain. BasT-inactivated mutant cells are missing a membrane protein radiolabelled with L-[methyl-3H]-methionine in wild-type cells, confirming that BasT is methylatable and membrane bound. Behavioural analysis of the basT mutant cells by capillary and chemical-in-plug assays demonstrates complete loss of chemotactic responses towards five (leucine, isoleucine, valine, methionine and cysteine) of the six attractant amino acids for Halobacterium salinarum, whereas they still respond to arginine. The volatile methyl group production assays also corroborate these findings and confirm that BasT signalling induces methyl group turnover. Our data identify BasT as the chemotaxis transducer protein for the branched chain amino acids leucine, isoleucine and valine as well as for methionine and cysteine. Thus, BasT and the arginine sensor Car cover the entire spectrum of chemotactic responses towards attractant amino acids in H. salinarum.     

Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells

Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-cross-linking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.     

Analysis of the proteins associated with platelet detergent resistant membranes

Proteomic studies have facilitated the identification of proteins associated with the detergent‐resistant membrane (DRM) fraction in a variety of cell types. Here, we have undertaken label‐free quantitative (LFQ) proteomic profiling of the proteins associated with detergent‐resistant plasma and internal membranes from resting and activated platelets. One hundred forty‐one proteins were identified and raw data is available via ProteomeXchange with identifier PXD002554. The proteins identified include a myriad of important platelet signaling and trafficking proteins including Rap1b, Src, SNAP‐23, syntaxin‐11, and members of the previously unattributed Ragulator complex. Mean LFQ intensities calculated across three technical replicates for the three biological donors revealed that several important platelet signaling proteins altered their detergent solubility upon activation, including GPIbα, GPIbβ, Src, and 14‐3‐3ζ. Altered detergent solubility for GPIbα, following activation using a variety of platelet agonists, was confirmed by immunoblotting and further coimmunoprecipitation experiments revealed that GPIbα forms a complex with 14‐3‐3ζ that shifts into DRMs following activation. Taken together, proteomic profiling of platelet DRMs allowed greater insight in the complex biology of both DRMs and platelets and will be a useful subproteome to study platelet‐related disease. All MS data have been deposited in the ProteomeXchange with identifier PXD002554 ( http://proteomecentral.proteomexchange.org/dataset/PXD002554 ).     

Differential quantitative proteome analysis of Escherichia coli grown on acetate versus glucose

Relative protein abundances of Escherichia coli MG1655 growing exponentially on minimal medium with acetate or glucose as the sole carbon source were investigated in a quantitative shotgun proteome analysis with TMT6‐plex isobaric tags. Peptides were separated by high resolution high/low pH 2D‐LC, using an optimized fraction pooling scheme followed by mass spectrometric analysis. Quantitative data were acquired for 2099 proteins covering 49% of the predicted E. coli proteins, showing system‐wide effects of growth conditions. In total, 507 proteins showed a fold change of at least 1.5 and 205 proteins changed by more than twofold. Significant differences in abundance were observed for most of the proteins in the central carbon metabolism and in proteins relevant for amino acid and protein synthesis, processing of environmental information and scavenging of a variety of alternate carbon sources. Periplasmic‐binding proteins were also more abundant on acetate, especially proteins involved in scavenging extracellular resources such as sugars. All MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD003863).     

Identification of N-terminal methionine in the precursor of immunoglobulin light chain. Initiation of translation of messenger ribonucleic acid in plants and animals.

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-321 mouse myeloma L (light) chain were labelled with [35S]methionine, [4,5-3H]leucine or [3-3H]serine, and were subjected to amino acid-sequence analyses. Over 95% of the total cell-free product was sequenced as one homogeneous protein, which corresponds to the precursor of the L-chain protein. In the precursor, 20 amino acid residues precede the N-terminus of the mature protein. This extra piece contains one methionine residue at the N-terminus, one serine residue at position 18, and six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13. The identification of methionine at the N-terminus of the precursor is in agreement with the evidence showing that unblocked methionine is the initiator residue for protein synthesis in eukaryotes. The absence of methionine at position 20, which precedes the N-terminal residue of the mature protein, suggests that myeloma cells synthesize the precursor. However, within the cell the precursor should be rapidly processed to the mature L chain, since precursor molecules have not yet been found in the intact animal. The abundance (30%) of leucine residues indicates that the extra-piece moiety is quite hydrophobic. The extra piece of the MOPC-321 L-chain precursor synthesized with the aid of the Krebs II ascites cell-free system is of identical size and it has the same leucine sequence [Schechter et al. (1975) Science 188, 160-162]. This indicates that cell-free systems derived from the plant and animal kingdom initiate mRNA translation from the same point. It is shown that the amino acid sequence of minute amounts of a highly labelled protein (0.1 pmol) can be faithfully determined in the presence of a large excess (over 2000 000-fold) of unrelated non-radioactive proteins.     

Proteomic identification of rainbow trout seminal plasma proteins

In the study, the combination of protein fractionation by 1DE and HPLC‐ESI‐MS/MS was used to characterize the rainbow trout seminal plasma proteome. Our results led to the creation of a catalogue of rainbow trout seminal plasma proteins (152 proteins) and significantly contributed to the current knowledge regarding the protein composition of fish seminal plasma. The major proteins of rainbow trout seminal plasma, such as transferrin, apolipoproteins, complement C3, serum albumin, and hemopexin‐, alpha‐1‐antiproteinase‐, and precerebellin‐like protein, were recognized as acute‐phase proteins (proteins that plasma concentration changes in response to inflammation). This study provides the basis for further functional studies of fish seminal plasma proteins, as well as for the identification of novel biomarkers for sperm quality. The MS data have been deposited in the ProteomeXchange with identifier PXD000306 ( http://proteomecentral.proteomexchange.org/dataset/PXD000306 ).     

Thermodynamic Effects of Noncoded and Coded Methionine Substitutions in Calmodulin

The methionine residues in the calcium (Ca²⁺) regulatory protein calmodulin (CaM) are structurally and functionally important. They are buried within the N- and C-domains of apo-CaM but become solvent-exposed in Ca²⁺-CaM, where they interact with numerous target proteins. Previous structural studies have shown that methionine substitutions to the noncoded amino acids selenomethionine, ethionine, or norleucine, or mutation to leucine do not impact the main chain structure of CaM. Here we used differential scanning calorimetry to show that these substitutions enhance the stability of both domains, with the largest increase in melting temperature (19-26°C) achieved with leucine or norleucine in the apo-C-domain. Nuclear magnetic resonance spectroscopy experiments also revealed the loss of a slow conformational exchange process in the Leu-substituted apo-C-domain. In addition, isothermal titration calorimetry experiments revealed considerable changes in the enthalpy and entropy of target binding to apo-CaM and Ca²⁺-CaM, but the free energy of binding was largely unaffected due to enthalpy-entropy compensation. Collectively, these results demonstrate that noncoded and coded methionine substitutions can be accommodated in CaM because of the structural plasticity of the protein. However, adjustments in side-chain packing and dynamics lead to significant differences in protein stability and the thermodynamics of target binding.     

Molecular cloning,expression, overproduction and characterization of human TRAIP Leucine zipper protein

The TRAIP interacting protein is known as a negative regulator of TNF-induced-nuclear factor, kappa-light-chain-enhancer of activated B cell (NF-κB) by direct interaction with the adaptor protein TRAF2, which inhibits the function of TRAF2 via the RINGCC domain protein. The TRAIP protein is composed of 469 amino acids with an N-terminal RING motif that is followed by a coiled coil (CC) and leucine zipper domain. TRAIP proteins are critical in programmed cell death, cell proliferation and differentiation, and embryonic development. The critical functions of TRAIP together with the molecular inhibitory mechanism effect of TRAIP have been reported by two different studies and have opened up new research into the field of TRAF biology. In this study, we designed different constructs of the Leucine zipper domain to find the over –expressed construct for further studies. We successfully cloned the C-terminal TRAIP containing the leucine zipper domain. In addition, we have over-expressed and purified the TRAIP LZ for their biochemical characterization.     

A network representation of protein structures: implications for protein stability

This study views each protein structure as a network of noncovalent connections between amino acid side chains. Each amino acid in a protein structure is a node, and the strength of the noncovalent interactions between two amino acids is evaluated for edge determination. The protein structure graphs (PSGs) for 232 proteins have been constructed as a function of the cutoff of the amino acid interaction strength at a few carefully chosen values. Analysis of such PSGs constructed on the basis of edge weights has shown the following: 1), The PSGs exhibit a complex topological network behavior, which is dependent on the interaction cutoff chosen for PSG construction. 2), A transition is observed at a critical interaction cutoff, in all the proteins, as monitored by the size of the largest cluster (giant component) in the graph. Amazingly, this transition occurs within a narrow range of interaction cutoff for all the proteins, irrespective of the size or the fold topology. And 3), the amino acid preferences to be highly connected (hub frequency) have been evaluated as a function of the interaction cutoff. We observe that the aromatic residues along with arginine, histidine, and methionine act as strong hubs at high interaction cutoffs, whereas the hydrophobic leucine and isoleucine residues get added to these hubs at low interaction cutoffs, forming weak hubs. The hubs identified are found to play a role in bringing together different secondary structural elements in the tertiary structure of the proteins. They are also found to contribute to the additional stability of the thermophilic proteins when compared to their mesophilic counterparts and hence could be crucial for the folding and stability of the unique three-dimensional structure of proteins. Based on these results, we also predict a few residues in the thermophilic and mesophilic proteins that can be mutated to alter their thermal stability.     

The role of free amino acids in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio

The present study investigated (1) the free amino acid (FAA) composition in semen of rainbow trout Oncorhynchus mykiss and carp Cyprinus carpio, (2) enzyme systems involved in amino acid metabolism and (3) the effect of amino acids on sperm viability under in vitro storage conditions. In the seminal plasma of O. mykiss, the main FAAs were arginine, glutamic acid, isoleucine, leucine, methionine and proline, in spermatozoa cysteine, arginine and methionine. In the seminal plasma of C. carpio, the main FAAs were alanine, arginine, cysteine, glutamic acid, histidine, leucine, lysine, methionine and proline, in spermatozoa arginine, glutamic acid, histidine, leucine and lysine. When spermatozoa were incubated for 48 h together with the seminal plasma, the quantitative amino acid pattern changed in both species indicating their metabolism. In spermatozoa and seminal plasma of O. mykiss and C. carpio, the following enzymes were found to be related to amino acid metabolism: transaminases (specific for alanine, aspartate, isoleucine and leucine), decarboxylases (specific for valine and lysine), glutamate dehydrogenase and α‐keto acid dehydrogenases (substrates: 3‐methyl‐2‐oxovaleric acid and 4‐methyl‐2‐oxovalerate). These data demonstrate that amino acid catabolism by transamination, decarboxylation and oxidative deamination can occur in semen of the two species. Also activity of methionine sulphoxide reductase was detected, an enzyme which reduces methionine sulphoxide to methionine. This reaction plays an important role in antioxidant defence. To determine the effect of FAAs on the sperm viability, C. carpio and O. mykiss spermatozoa were incubated in sperm motility inhibiting saline solution containing different amino acids. Methionine had a positive effect on the sperm viability in both species. Taken together this result with the in vivo occurrence of methionine and of methionine reductase in semen, it can be assumed that this amino acid plays an important role in antioxidant defence. Also isoleucine in O. mykiss and leucine in C. carpio had a positive effect on sperm viability. As seminal plasma and spermatozoa of the two species exhibit enzyme activities to catabolize leucine and isoleucine, they might serve as additional energy resources especially during prolonged incubation and storage periods.     

Defining the proteome of human iris,ciliary body,retinal pigment epithelium,and choroid

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS‐PAGE. After in‐gel digestion, peptides were analyzed using LC‐MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false‐positive rates of <0.1% and <1%, respectively. Forty‐three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four “missing proteins” were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.     

Proteome and carbon flux analysis of Pseudomonas aeruginosa clinical isolates from different infection sites

Pseudomonas aeruginosa is known as opportunistic pathogen frequently isolated from different infection sites. To investigate the expression rates of P. aeruginosa proteins commonly expressed by different clinical isolates, absolute protein quantities were determined employing a gel‐free and data‐independent LC‐IMS^E approach. Moreover, the metabolic diversity of these isolates was investigated by ¹³C‐metabolic flux analyses. 812 proteins were reproducibly identified and absolutely quantified for the reference strain P. aeruginosa PAO1, 363 of which were also identified and relatively quantified in all isolates. Whilst the majority of these proteins were expressed in constant amounts, expression rates of 42 proteins were highly variable between the isolates. Notably, the outer membrane protein OprH and the response regulator PhoP were strongly expressed in burned wounds isolates compared to lung/urinary tract isolates. Moreover, proteins involved in iron/amino acids uptake were found to be highly abundant in urinary tract isolates. The fluxome data revealed a conserved glycolysis, and a niche‐specific divergence in fluxes through the glyoxylate shunt and the TCA cycle among the isolates. The integrated proteome/fluxome analysis did not indicate straightforward correlation between the protein amount and flux, but rather points to additional layers of regulation that mediate metabolic adaption of P. aeruginosa to different host environments. All MS data have been deposited in the ProteomeXchange with identifier PXD002373 ( http://proteomecentral.proteomexchange.org/dataset/PXD002373 ).     

Analysis of the interactome of ribosomal protein S19 mutants

Diamond–Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one‐fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild‐type protein one, using the same functional proteomic approach that we employed to characterize RPS19 interactome. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. Results presented in this paper highlight the striking differences between the interactomes of wild‐type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control. The data have been deposited to the ProteomeXchange with identifier PXD000640 ( http://proteomecentral.proteomexchange.org/dataset/PXD000640 ).