Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take ...advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning–based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the
proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.
Old enzymes versus new herbicides Hendrickson, Tamara L.
Journal of biological chemistry/The Journal of biological chemistry,
05/2018, Letnik:
293, Številka:
20
Journal Article
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Odprti dostop
The introduction of manmade chemicals, including the herbicide atrazine, into the environment has led to the emergence of microorganisms with new biodegradation pathways. Esquirol et al. demonstrate ...that the AtzE enzyme catalyzes a central step in atrazine degradation and that expression of AtzE requires coexpression of the small protein AtzG. Remarkably, AtzG and AtzE appear to have evolved from GatC and GatA, components of an ancient enzyme involved in indirect tRNA aminoacylation, providing an elegant demonstration of metabolic repurposing.
Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, post-translational modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces cerevisiae GPI-T is composed of five ...membrane-bound subunits: Gpi8, Gaa1, Gpi16, Gpi17, and Gab1. GPI-T has been recalcitrant to in vitro structure and function studies because of its complexity and membrane-solubility. Furthermore, a reliable, quantitative, in vitro assay for this important post-translational modification has remained elusive despite its discovery more than three decades ago.Three recent reports describe the structure of GPI-T from S. cerevisiae and humans, shedding critical light on this important enzyme and offering insight into the functions of its different subunits. Here, we present the purification and characterization of a truncated soluble GPI-T heterotrimer complex (Gpi823–306, Gaa150–343, and Gpi1620–551) without transmembrane domains. Using this simplified heterotrimer, we report the first quantitative method to measure GPI-T activity in vitro and demonstrate that this soluble, minimalistic GPI-T retains transamidase activity. These results contribute to our understanding of how this enzyme is organized and functions, and provide a method to screen potential GPI-T inhibitors.
Abstract
Cancer is second only to heart disease as a cause of death in the US, with a further negative economic impact on society. Over the past decade, details have emerged which suggest that ...different glycosylphosphatidylinositol (GPI)-anchored proteins are fundamentally involved in a range of cancers. This post-translational glycolipid modification is introduced into proteins via the action of the enzyme GPI transamidase (GPI-T). In 2004, PIG-U, one of the subunits of GPI-T, was identified as an oncogene in bladder cancer, offering a direct connection between GPI-T and cancer. GPI-T is a membrane-bound, multi-subunit enzyme that is poorly understood, due to its structural complexity and membrane solubility. This review is divided into three sections. First, we describe our current understanding of GPI-T, including what is known about each subunit and their roles in the GPI-T reaction. Next, we review the literature connecting GPI-T to different cancers with an emphasis on the variations in GPI-T subunit over-expression. Finally, we discuss some of the GPI-anchored proteins known to be involved in cancer onset and progression and that serve as potential biomarkers for disease-selective therapies. Given that functions for only one of GPI-T's subunits have been robustly assigned, the separation between healthy and malignant GPI-T activity is poorly defined.
Most bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNA
and tRNA
. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction ...in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in
, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in
impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type
or one of two
mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.
Most bacteria use a two-step indirect pathway to aminoacylate tRNA
and tRNA
, despite the fact that the indirect pathway consumes more energy and is error prone. We have previously shown that the higher protein synthesis errors from this indirect pathway in mycobacteria allow adaptation to hostile environments such as antibiotic treatment through generation of novel alternate proteins not coded by the genome. However, the precise mechanisms of how translational fidelity is tuned were not known. Here, we biochemically and biophysically characterize the critical enzyme of the Mycobacterium tuberculosis indirect pathway, GatCAB, as well as two mutant enzymes previously identified from clinical isolates that were associated with increased mistranslation. We show that the mutants dysregulate the pathway via destabilizing the enzyme complex. Importantly, increasing stability improves translational fidelity in both wild-type and mutant bacteria, demonstrating a mechanism by which mycobacteria may tune mistranslation rates.
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•Many bacteria are missing asparaginyl-tRNA and/or glutaminyl-tRNA synthetase.•Asn-tRNAAsn and Gln-tRNAGln are produced in a two-step, indirect pathway.•GatCAB uses an unusually ...hydrophilic ammonia tunnel to transport ammonia between active sites.•A large complex called the Asn-transamidosome sequesters Asp-tRNAAsn from the ribosome.•Indirect tRNA aminoacylation can induce beneficial mistranslation under stress.
The fact that most bacteria do not contain a full set of aminoacyl-tRNA synthetases (aaRS) is often underappreciated. In the absence of asparaginyl-tRNA and/or glutaminyl-tRNA synthetase (AsnRS and GlnRS), Asn-tRNAAsn and/or Gln-tRNAGln are produced by an indirect tRNA aminoacylation pathway that relies on misacylation of these two tRNAs by two different misacylating aaRSs, followed by transamidation by an amidotransferase (GatCAB in bacteria). This review highlights the central importance of indirect tRNA aminoacylation to accurate protein translation, mechanistic peculiarities that appear to be unique to this system, and the newly recognized connection between indirect tRNA aminoacylation and mistranslation as a strategy used by bacteria to respond to environmental stressors like antibiotics.
The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their ...metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.
The aminoacyl-tRNA synthetases (aaRSs) are well established as the translators of the genetic code, because their products, the aminoacyl-tRNAs, read codons to translate messenger RNAs into proteins. ...Consequently, deleterious errors by the aaRSs can be transferred into the proteome via misacylated tRNAs. Nevertheless, many microorganisms use an indirect pathway to produce Asn-tRNA
via Asp-tRNA
. This intermediate is produced by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) that has retained its ability to also generate Asp-tRNA
. Here we report the discovery that ND-AspRS and its discriminating counterpart, AspRS, are also capable of specifically producing Glu-tRNA
, without producing misacylated tRNAs like Glu-tRNA
, Glu-tRNA
, or Asp-tRNA
, thus maintaining the fidelity of the genetic code. Consequently, bacterial AspRSs have glutamyl-tRNA synthetase-like activity that does not contaminate the proteome via amino acid misincorporation.
Glycosylphosphatidylinositol transamidase (GPI-T) catalyzes the post-translational addition of the GPI anchor to the C-terminus of some proteins. In most eukaryotes, Gpi8, the active site subunit of ...GPI-T, is part of a hetero-pentameric complex containing Gpi16, Gaa1, Gpi17, and Gab1. Gpi8, Gaa1, and Gpi16 co-purify as a heterotrimer from Saccharomyces cerevisiae, suggesting that they form the core of the GPI-T. Details about the assembly and organization of these subunits have been slow to emerge. We have previously shown that the soluble domain of S. cerevisiae Gpi8 (Gpi823-306) assembles as a homodimer, similar to the caspases with which it shares weak sequence homology (Meitzler, J. L. et al., 2007). Here we present the characterization of a complex between the soluble domains of Gpi8 and Gaa1. The complex between GST-Gpi823-306 (α) and His6-Gaa150-343 (β) was characterized by native gel analysis and size exclusion chromatography (SEC) and results are most consistent with an α2β2 stoichiometry. These results demonstrate that Gpi8 and Gaa1 interact specifically without a requirement for other subunits, bring us closer to determining the stoichiometry of the core subunits of GPI-T, and lend further credence to the hypothesis that these three subunits assemble into a dimer of a trimer.
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•The soluble domains of Gpi823-306 and Gaa150-343 form a stable, discrete complex.•The Gpi823-306:Gaa150-343 complex adopts an α2β2 stoichiometry.•The Gaa150-343 monomer and homodimer were not observed in the absence of Gpi823-306.•Truncation of a conserved face of Gaa150-343 did not disrupt assembly of the Gpi823-306:Gaa150-343 complex.
This paper covers direct sub‐atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not ...based on the high‐energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas‐phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix‐assisted ionization (MAI) and solvent‐assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.