Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell–mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell–mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1–triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1–targeting therapeutic approaches.     

Global analysis of protein-protein interactions reveals multiple CYP2E1-reductase complexes

Although a single binary functional complex between cytochrome P450 (P450 or CYP for a specific isoform) and cytochrome P450 reductase (CPR) has been generally accepted in the literature, this simple model failed to explain the experimentally observed catalytic activity of recombinant CYP2E1 in dependence on the total concentration of the added CPR-K56Q mutant. Our rejection of the simplest 1:1 binding model was based on two independent lines of experimental evidence. First, under the assumption of the 1:1 binding model, separate analyses of titration curves obtained while varying either P450 or CPR concentrations individually produced contradictory results. Second, an asymmetric Job plot suggested the existence of higher order molecular complexes. To identify the most probable complexation mechanism, we generated a comprehensive data set where the concentrations of both P450 and P450 were varied simultaneously, rather than one at a time. The resulting two-dimensional data were globally fit to 32 candidate mechanistic models, involving the formation of binary, ternary, and quaternary P450.CPR complexes, in the absence or presence or P450 and CPR homodimers. Of the 32 candidate models (mechanisms), two models were approximately equally successful in explaining our experimental data. The first plausible model involves the binary complex P450.CPR, the quaternary complex (P450)2.(CPR)2, and the homodimer (P450)2. The second plausible model additionally involves a weakly bound ternary complex (P450)2.CPR. Importantly, only the binary complex P450.CPR seems catalytically active in either of the two most probable mechanisms.     

Single-molecule analysis reveals three phases of DNA degradation by an exonuclease

λ exonuclease degrades one strand of duplex DNA in the 5'-to-3' direction to generate a 3' overhang required for recombination. Its ability to hydrolyze thousands of nucleotides processively is attributed to its ring structure, and most studies have focused on the processive phase. Here we have used single-molecule fluorescence resonance energy transfer (FRET) to reveal three phases of λ exonuclease reactions: the initiation, distributive and processive phases. The distributive phase comprises early reactions in which the 3' overhang is too short to stably engage with the enzyme. A mismatched base is digested one-fifth as quickly as a Watson-Crick-paired base, and multiple concatenated mismatches have a cooperatively negative effect, highlighting the crucial role of base pairing in aligning the 5' end toward the active site. The rate-limiting step during processive degradation seems to be the post-cleavage melting of the terminal base pair. We also found that an escape from a known pausing sequence requires enzyme backtracking.     

Optimal transport analysis reveals trajectories in steady-state systems

Understanding how cells change their identity and behaviour in living systems is an important question in many fields of biology. The problem of inferring cell trajectories from single-cell measurements has been a major topic in the single-cell analysis community, with different methods developed for equilibrium and non-equilibrium systems (e.g. haematopoeisis vs. embryonic development). We show that optimal transport analysis, a technique originally designed for analysing time-courses, may also be applied to infer cellular trajectories from a single snapshot of a population in equilibrium. Therefore, optimal transport provides a unified approach to inferring trajectories that is applicable to both stationary and non-stationary systems. Our method, StationaryOT, is mathematically motivated in a natural way from the hypothesis of a Waddington’s epigenetic landscape. We implement StationaryOT as a software package and demonstrate its efficacy in applications to simulated data as well as single-cell data from Arabidopsis thaliana root development.     

Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.     

Regulation of sugar transport via the multiple sugar metabolism operon of Streptococcus mutans by the phosphoenolpyruvate phosphotransferase system.

In this report, we provide evidence that the transport of sugars in Streptococcus mutans via the multiple sugar metabolism system is regulated by the phosphoenolpyruvate phosphotransferase system. A ptsI-defective mutant (DC10), when grown on the multiple sugar metabolism system substrate raffinose, exhibited reduced growth, transport, and glycolytic activity with raffinose relative to the parent strain BM71. Inhibition of [3H]raffinose uptake was also observed in both BM71 and DC10 with increasing concentrations of glucose and the glucose analogs alpha-methyl glucoside and 2-deoxyglucose.     

Distinct mechanisms for Ctr1-mediated copper and cisplatin transport

The Ctr1 family of integral membrane proteins is necessary for high affinity copper uptake in eukaryotes. Ctr1 is also involved in cellular accumulation of cisplatin, a platinum-based anticancer drug. Although the physiological role of Ctr1 has been revealed, the mechanism of action of Ctr1 remains to be elucidated. To gain a better understanding of Ctr1-mediated copper and cisplatin transport, we have monitored molecular dynamics and transport activities of yeast Saccharomyces cerevisiae Ctr1 and its mutant alleles. Co-expression of functional Ctr1 monomers fused with either cyan or yellow fluorescent protein resulted in fluorescence resonance energy transfer (FRET), which is consistent with multimer assembly of Ctr1. Copper near the K(m) value of Ctr1 enhanced FRET in a manner that correlated with cellular copper transport. In vitro cross-linking of Ctr1 confirmed that copper-induced FRET reflects conformational changes within pre-existing Ctr1 complexes. FRET assays in membrane-disrupted cells and protein extracts showed that intact cell structure is necessary for Ctr1 activity. Despite Ctr1-dependent cellular accumulation, cisplatin did not change Ctr1 FRET nor did it attenuate copper-induced FRET. A Ctr1 allele defective in copper transport enhanced cellular cisplatin accumulation. N-terminal methionine-rich motifs that are dispensable for copper transport play a critical role for cisplatin uptake. Taken together, our data reveal functional roles for structural remodeling of the Ctr1 multimeric complex in copper transport and suggest distinct mechanisms employed by Ctr1 for copper and cisplatin transport.     

PepT1-mediated fMLP transport induces intestinal inflammation in vivo

In the presentstudy, the effect of H⁺/peptide transporter(PepT1)-mediatedN-formylmethionyl-leucyl-phenylalanine (fMLP)transport on inflammation in vivo in the rat small intestine, whichexpresses high PepT1 levels, and in the rat colon, which does notexpress PepT1, were investigated using myeloperoxidase (MPO) activity and histological analysis. We found that 10 µM fMLP perfusion in thejejunum for 4 h significantly increased MPO activity and alteredthe architecture of jejunal villi. In contrast, 10 µM fMLP perfusionin the colon for 4 h did not induce any inflammation. In addition,we have shown that 50 mM Gly-Gly alone did not affect basal MPOactivity but completely inhibited the MPO activity induced by 10 µMfMLP in the jejunum. Together, these experiments demonstrate that1) the differential expression of PepT1 between the small intestine and the colon plays an important role inepithelial-neutrophil interactions and 2) the inhibition offMLP uptake by jejunal epithelial cells (expressing PepT1) reduces theneutrophil ability to move across the epithelium, in agreement with ourpreviously published in vitro study. This report constitutes the firstin vivo study showing the implication of a membrane transporter (PepT1)in intestinal inflammation.

     

Multilocus sequence analysis reveals multiple symbiovars within Mesorhizobium species

The genus Mesorhizobium includes species nodulating several legumes, such as chickpea, which has a high agronomic importance. Chickpea rhizobia were originally described as either Mesorhizobium ciceri or M. mediterraneum. However, rhizobia able to nodulate chickpea have been shown to belong to several different species within the genus Mesorhizobium. The present study used a multilocus sequence analysis approach to infer a high resolution phylogeny of the genus Mesorhizobium and to confirm the existence of a new chickpea nodulating genospecies. The phylogenetic structure of the Mesorhizobium clade was evaluated by sequence analysis of the 16S rRNA gene, ITS region and the five core genes atpD, dnaJ, glnA, gyrB, and recA. Phylogenies obtained with the different genes are in overall good agreement and a well-supported, almost fully resolved, phylogenetic tree was obtained using the combined data. Our phylogenetic analyses of core genes sequences and their comparison with the symbiosis gene nodC, corroborate the existence of one new chickpea Mesorhizobium genospecies and one new symbiovar, M. opportunistum sv. ciceri. Furthermore, our results show that symbiovar ciceri spreads over six species of mesorhizobia. To our knowledge this study shows the most complete Mesorhizobium multilocus phylogeny to date and contributes to the understanding of how a symbiovar may be present in different species.     

A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism.

An 11-kilobase gene region of Streptococcus mutans has been identified which contains eight contiguous genes involved with the uptake and metabolism of multiple sugars (the msm system). Sequence analysis of this region indicates that several of these genes specify proteins with strong homology to components of periplasmic binding protein-dependent transport systems of Gram-negative bacteria. Additionally, this operon is controlled by a regulatory gene (msmR) that acts as a positive effector. The proteins specified by the structural genes of the msm operon include alpha-galactosidase (aga), a "periplasmic-like" sugar-binding protein (msmE), two membrane proteins (msmF, msmG), sucrose phosphorylase (gtfA), an ATP-binding protein (msmK), and dextran glucosidase (dexB). Insertional inactivation of each of these genes along with uptake data indicate that this system is responsible for the uptake of melibiose, raffinose, and isomaltotriose and the metabolism of melibiose, sucrose, and isomaltosaccharides.     

Phylogenetic analysis reveals multiple introductions of Cynodon species in Australia

The distinction between native and introduced flora within isolated land masses presents unique challenges. The geological and colonisation history of Australia, the world's largest island, makes it a valuable system for studying species endemism, introduction, and phylogeny. Using this strategy we investigated Australian cosmopolitan grasses belonging to the genus Cynodon. While it is believed that seven species of Cynodon are present in Australia, no genetic analyses have investigated the origin, diversity and phylogenetic history of Cynodon within Australia. To address this gap, 147 samples (92 from across Australia and 55 representing global distribution) were sequenced for a total of 3336bp of chloroplast DNA spanning six genes. Data showed the presence of at least six putatively introduced Cynodon species (C. transvaalensis, C. incompletus, C. hirsutus, C. radiatus, C. plectostachyus and C. dactylon) in Australia and suggested multiple recent introductions. C. plectostachyus, a species often confused with C. nlemfuensis, was not previously considered to be present in Australia. Most significantly, we identified two common haplotypes that formed a monophyletic clade diverging from previously identified Cynodon species. We hypothesise that these two haplotypes may represent a previously undescribed species of Cynodon. We provide further evidence that two Australian native species, Brachyachne tenella and B. convergens belong in the genus Cynodon and, therefore, argue for the taxonomic revision of the genus Cynodon.     

ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins

Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.     

Solution single-vesicle assay reveals PIP2-mediated sequential actions of synaptotagmin-1 on SNAREs

Synaptotagmin-1 (Syt1) is a major Ca(2+) sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t-) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single-molecule technique, alternating-laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca(2+), Syt1 mediates vesicle docking by directly binding to t-SNARE/phosphatidylinositol 4,5-biphosphate (PIP(2)) complex and increases the docking rate by 10(3) times. Second, synaptobrevin-2 binding to t-SNARE displaces Syt1 from SNAREpin. Third, with Ca(2+), Syt1 rebinds to SNAREpin, which again requires PIP(2). Thus without Ca(2+), Syt1 may bring vesicles to the plasma membrane in proximity via binding to t-SNARE/PIP(2) to help SNAREpin formation and then, upon Ca(2+) influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway.