Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion.

T cell exhaustion is linked to persistent antigen exposure and perturbed activation events, correlating with poor disease prognosis. Tumor-mediated T cell exhaustion is well documented; however, how the nutrient-deprived tumor niche affects T cell receptor (TCR) activation is largely unclear. We show that methionine metabolism licenses optimal TCR signaling by regulating the protein arginine methylome, and limiting methionine availability during early TCR signaling promotes subsequent T cell exhaustion. We discovered a novel arginine methylation of a Ca 2+ -activated potassium transporter, KCa3.1, prevention of which results in increased Ca 2+ -mediated NFAT1 activation, NFAT1 promoter occupancy, and T cell exhaustion. Furthermore, methionine supplementation reduces nuclear NFAT1 in tumor-infiltrating T cells and augments their anti-tumor activity. These findings demonstrate metabolic regulation of T cell exhaustion determined during TCR engagement.

Cells alter their tRNA abundance to selectively regulate protein synthesis during stress conditions.

Decoding the information in mRNA during protein synthesis relies on tRNA adaptors, the abundance of which can affect the decoding rate and translation efficiency. To determine whether cells alter tRNA abundance to selectively regulate protein expression, we quantified changes in the abundance of individual tRNAs at different time points in response to diverse stress conditions in Saccharomyces cerevisiae We found that the tRNA pool was dynamic and rearranged in a manner that facilitated selective translation of stress-related transcripts. Through genomic analysis of multiple data sets, stochastic simulations, and experiments with designed sequences of proteins with identical amino acids but altered codon usage, we showed that changes in tRNA abundance affected protein expression independently of factors such as mRNA abundance. We suggest that cells alter their tRNA abundance to selectively affect the translation rates of specific transcripts to increase the amounts of required proteins under diverse stress conditions.

Probing Gαi1 protein activation at single-amino acid resolution.

We present comprehensive maps at single-amino acid resolution of the residues stabilizing the human Gαi1 subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of Gαi1 and the rhodopsin-Gαi1 complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices α1 and α5 pack against strands ß1-ß3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between α5 and strands ß4-ß6. Key residues in this cluster are Y320, which is crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix α1, caused by rearrangement of this activation cluster, leads to the weakening of the interdomain interface and release of GDP.

p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients.

Structured and disordered facets of the GPCR fold.

The seven-transmembrane (7TM) helix fold of G-protein coupled receptors (GPCRs) has been adapted for a wide variety of physiologically important signaling functions. Here, we discuss the diversity in the structured and disordered regions of GPCRs based on the recently published crystal structures and sequence analysis of all human GPCRs. A comparison of the structures of rhodopsin-like receptors (class A), secretin-like receptors (class B), metabotropic receptors (class C) and frizzled receptors (class F) shows that the relative arrangement of the transmembrane helices is conserved across all four GPCR classes although individual receptors can be activated by ligand binding at varying positions within and around the transmembrane helical bundle. A systematic analysis of GPCR sequences reveals the presence of disordered segments in the cytoplasmic side, abundant post-translational modification sites, evidence for alternative splicing and several putative linear peptide motifs that have the potential to mediate interactions with cytosolic proteins. While the structured regions permit the receptor to bind diverse ligands, the disordered regions appear to have an underappreciated role in modulating downstream signaling in response to the cellular state. An integrated paradigm combining the knowledge of structured and disordered regions is imperative for gaining a holistic understanding of the GPCR (un)structure-function relationship.

Structural polymorphism in the N-terminal oligomerization domain of NPM1.

Nucleophosmin (NPM1) is a multifunctional phospho-protein with critical roles in ribosome biogenesis, tumor suppression, and nucleolar stress response. Here we show that the N-terminal oligomerization domain of NPM1 (Npm-N) exhibits structural polymorphism by populating conformational states ranging from a highly ordered, folded pentamer to a highly disordered monomer. The monomer-pentamer equilibrium is modulated by posttranslational modification and protein binding. Phosphorylation drives the equilibrium in favor of monomeric forms, and this effect can be reversed by Npm-N binding to its interaction partners. We have identified a short, arginine-rich linear motif in NPM1 binding partners that mediates Npm-N oligomerization. We propose that the diverse functional repertoire associated with NPM1 is controlled through a regulated unfolding mechanism signaled through posttranslational modifications and intermolecular interactions.

Chemogenomics and biotechnology.

A robust knowledge of the interactions between small molecules and specific proteins aids the development of new biotechnological tools and the identification of new drug targets, and can lead to specific biological insights. Such knowledge can be obtained through chemogenomic screens. In these screens, each small molecule from a chemical library is applied to each cell type from a library of cells, and the resulting phenotypes are recorded. Chemogenomic screens have recently become very common and will continue to generate large amounts of data. The interpretation of this data will occupy biologists and chemists alike for some time to come. This review discusses methods for the acquisition and interpretation of chemogenomic data, in addition to possible applications of chemogenomics in biotechnology.

Evolutionary dynamics of prokaryotic transcriptional regulatory networks.

The structure of complex transcriptional regulatory networks has been studied extensively in certain model organisms. However, the evolutionary dynamics of these networks across organisms, which would reveal important principles of adaptive regulatory changes, are poorly understood. We use the known transcriptional regulatory network of Escherichia coli to analyse the conservation patterns of this network across 175 prokaryotic genomes, and predict components of the regulatory networks for these organisms. We observe that transcription factors are typically less conserved than their target genes and evolve independently of them, with different organisms evolving distinct repertoires of transcription factors responding to specific signals. We show that prokaryotic transcriptional regulatory networks have evolved principally through widespread tinkering of transcriptional interactions at the local level by embedding orthologous genes in different types of regulatory motifs. Different transcription factors have emerged independently as dominant regulatory hubs in various organisms, suggesting that they have convergently acquired similar network structures approximating a scale-free topology. We note that organisms with similar lifestyles across a wide phylogenetic range tend to conserve equivalent interactions and network motifs. Thus, organism-specific optimal network designs appear to have evolved due to selection for specific transcription factors and transcriptional interactions, allowing responses to prevalent environmental stimuli. The methods for biological network analysis introduced here can be applied generally to study other networks, and these predictions can be used to guide specific experiments.

Evolution of transcription factors and the gene regulatory network in Escherichia coli.

The most detailed information presently available for an organism's transcriptional regulation network is that for the prokaryote Escherichia coli. In order to gain insight into the evolution of the E.coli regulatory network, we analysed information obtainable for the domains and protein families of the transcription factors and regulated genes. About three-quarters of the 271 transcription factors we identified are two-domain proteins, consisting of a DNA-binding domain along with a regulatory domain. The regulatory domains mainly bind small molecules. Many groups of transcription factors have identical domain architectures, and this implies that roughly three-quarters of the transcription factors have arisen as a consequence of gene duplication. In contrast, there is little evidence of duplication of regulatory regions together with regulated genes or of transcription factors together with regulated genes. Thirty-eight, out of the 121 transcription factors for which one or more regulated genes are known, regulate other transcription factors. This amplification effect, as well as large differences between the numbers of genes directly regulated by transcription factors, means that there are about 10 global regulators which each control many more genes than the other transcription factors.

Did the loss of sigma factors initiate pseudogene accumulation in M. leprae?

Pseudogenes are non-functional regions in the genome that have arisen as a consequence of accumulating mutations that either result in the premature termination of proteins during protein synthesis or the disruption of transcription. There have been various discussions of the origins of pseudogenes and the models for their formation, but there has been little input on how pseudogenes could have accumulated in an organism. In this brief communication, I propose a two-step model for the accretion of pseudogenes in the Mycobacterium leprae genome, triggered by the loss of different sets of sigma factors at different time points during the course of evolution.

Functional determinants of transcription factors in Escherichia coli: protein families and binding sites.

DNA-binding transcription factors regulate the expression of genes near to where they bind. These factors can be activators or repressors of transcription, or both. Thus, a fundamental question is what determines whether a transcription factor acts as an activator or a repressor? Previous research into this question found that a protein's regulatory function is determined by one or more of the following factors: protein-protein contacts, position of the DNA-binding domain in the protein primary sequence, altered DNA structure, and the position of its binding site on the DNA relative to the transcription start site. Although there are many aspects specific to different transcription factors, in this work we demonstrate that, in general, in the prokaryote Escherichia coli, a transcription factor's protein family is not indicative of its regulatory function, but the position of its binding site on the DNA is.

A C-H triplebond O hydrogen bond stabilized polypeptide chain reversal motif at the C terminus of helices in proteins.

The serendipitous observation of a C-H cdots, three dots, centered O hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C-H triplebond O interaction between the T-4 C(alpha)H and T+1 Cz doublebond O group (C triplebond O< or =3.5A) becomes possible only when the T+1 residue adopts an extended beta conformation (T is defined as the helix terminating residue adopting an alpha(L) conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational preferences at positions T-4, T, and T+1 determined. A marked preference for residues like Ser, Glu and Gln is observed at T-4 position with the motif being further stabilized by the formation of a side-chain-backbone O triplebond H-N hydrogen bond involving the side-chain of residue T-4 and the N-H group of residue T+3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in beta conformation. In a majority of these cases, the succeeding beta strand lies approximately antiparallel with the helix, suggesting that the backbone C-H triplebond O interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C-H cdots, three dots, centered O hydrogen bonds between (T-4) C(alpha)H triplebond O (T+1) and (T-8) C(alpha)H triplebondC doublebond O (T+3).

DOLOP--database of bacterial lipoproteins.

UNLABELLED: Bacterial lipoproteins and lipid modification are gaining importance owing to their essential nature, roles in pathogenesis and interesting commercial applications. We have created an exclusive knowledge base for bacterial lipoproteins by processing information from 510 entries to provide a list of 199 distinct lipoproteins with relevant links to molecular details. Features include functional classification, predictive algorithm for query sequences, primary sequence analysis and lists of predicted lipoproteins from 43 completed bacterial genomes along with interactive information exchange facility. AVAILABILITY: The website called Database Of bacterial LipOProteins (DOLOP) is available at http://www.mrc-lmb.cam.ac.uk/genomes/dolop along with additional information on the biosynthetic pathway, supplementary material and other related figures.