scholarly journals N-glycan chitobiose core biosynthesis by Agl24 strengthens the hypothesis of an archaeal origin of the eukaryal N-glycosylation

2021 ◽  
Author(s):  
Benjamin H. Meyer ◽  
Ben A. Wagstaff ◽  
Panagiotis S. Adam ◽  
Sonja-Verena Albers ◽  
Helge C. Dorfmueller
Keyword(s):  
Posttranslational Modifications ◽  
Biochemical Characterization ◽  
Similar Process ◽  
List Type ◽  
Histidine Residue ◽  
Functional Importance ◽  
Domains Of Life ◽  
Distant Relation ◽  
Identification And Characterization

AbstractProtein N-glycosylation is the most common posttranslational modifications found in all three domains of life. The crenarchaeal N-glycosylation begins with the synthesis of a lipid-linked chitobiose core structure, identical to that in eukaryotes. Here, we report the identification of a thermostable archaeal beta-1,4-N-acetylglucosaminyltransferase, named archaeal glycosylation enzyme 24 (Agl24), responsible for the synthesis of the N-glycan chitobiose core. Biochemical characterization confirmed the function as an inverting β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase. Substitution of a conserved histidine residue, found also in the eukaryotic and bacterial homologs, demonstrated its functional importance for Agl24. Furthermore, bioinformatics and structural modeling revealed strong similarities between Agl24 and both the eukaryotic Alg14/13 and a distant relation to the bacterial MurG, which catalyze the identical or a similar process, respectively. Our data, complemented by phylogenetic analysis of Alg13 and Alg14, revealed similar sequences in Asgardarchaeota, further supporting the hypothesis that the Alg13/14 homologs in eukaryotes have been acquired during eukaryogenesis.HighlightsFirst identification and characterization of a thermostable β-D-GlcNAc-(1→4)-α-D-GlcNAc-diphosphodolichol glycosyltransferase (GT family 28) in Archaea.A highly conserved histidine, within a GGH motif in Agl24, Alg14, and MurG, is essential for function of Agl24.Agl24-like homologs are broadly distributed among Archaea.The eukaryotic Alg13 and Alg14 are closely related to the Asgard homologs, suggesting their acquisition during eukaryogenesis.

scholarly journals Identification and Characterization of Thermostable Y-Family DNA Polymerases η, ι, κ and Rev1 From a Lower Eukaryote, Thermomyces lanuginosus

2021 ◽  
Vol 8 ◽  
Author(s):  
Alexandra Vaisman ◽  
John P. McDonald ◽  
Mallory R. Smith ◽  
Sender L. Aspelund ◽  
Thomas C. Evans ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Dna Polymerases ◽  
Biochemical Properties ◽  
Thermomyces Lanuginosus ◽  
Thermophilic Fungi ◽  
Domains Of Life ◽  
Y Family ◽  
Identification And Characterization ◽  
Lower Eukaryote

Y-family DNA polymerases (pols) consist of six phylogenetically separate subfamilies; two UmuC (polV) branches, DinB (pol IV, Dpo4, polκ), Rad30A/POLH (polη), and Rad30B/POLI (polι) and Rev1. Of these subfamilies, DinB orthologs are found in all three domains of life; eubacteria, archaea, and eukarya. UmuC orthologs are identified only in bacteria, whilst Rev1 and Rad30A/B orthologs are only detected in eukaryotes. Within eukaryotes, a wide array of evolutionary diversity exists. Humans possess all four Y-family pols (pols η, ι, κ, and Rev1), Schizosaccharomyces pombe has three Y-family pols (pols η, κ, and Rev1), and Saccharomyces cerevisiae only has polη and Rev1. Here, we report the cloning, expression, and biochemical characterization of the four Y-family pols from the lower eukaryotic thermophilic fungi, Thermomyces lanuginosus. Apart from the expected increased thermostability of the T. lanuginosus Y-family pols, their major biochemical properties are very similar to properties of their human counterparts. In particular, both Rad30B homologs (T. lanuginosus and human polɩ) exhibit remarkably low fidelity during DNA synthesis that is template sequence dependent. It was previously hypothesized that higher organisms had acquired this property during eukaryotic evolution, but these observations imply that polι originated earlier than previously known, suggesting a critical cellular function in both lower and higher eukaryotes.

scholarly journals Mycobacteriophages as Incubators for Intein Dissemination and Evolution

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Danielle S. Kelley ◽  
Christopher W. Lennon ◽  
Marlene Belfort ◽  
Olga Novikova ◽  
Keyword(s):  
Biochemical Characterization ◽  
Bioinformatic Analysis ◽  
Posttranslational Regulation ◽  
Homing Endonucleases ◽  
Nucleic Acid Binding ◽  
Active Centers ◽  
Functional Sites ◽  
Domains Of Life ◽  
Self Splicing

ABSTRACT Inteins are self-splicing protein elements that are mobile at the DNA level and are sporadically distributed across microbial genomes. Inteins appear to be horizontally transferred, and it has been speculated that phages may play a role in intein distribution. Our attention turns to mycobacteriophages, which infect mycobacteria, where both phage and host harbor inteins. Using bioinformatics, mycobacteriophage genomes were mined for inteins. This study reveals that these mobile elements are present across multiple mycobacteriophage clusters and are pervasive in certain genes, like the large terminase subunit TerL and a RecB-like nuclease, with the majority of intein-containing genes being phage specific. Strikingly, despite this phage specificity, inteins localize to functional motifs shared with bacteria, such that intein-containing genes have similar roles, like hydrolase activity and nucleic acid binding, indicating a global commonality among intein-hosting proteins. Additionally, there are multiple insertion points within active centers, implying independent invasion events, with regulatory implications. Several phage inteins were shown to be splicing competent and to encode functional homing endonucleases, important for mobility. Further, bioinformatic analysis supports the potential for phages as facilitators of intein movement among mycobacteria and related genera. Analysis of catalytic intein residues finds the highly conserved penultimate histidine inconsistently maintained among mycobacteriophages. Biochemical characterization of a noncanonical phage intein shows that this residue influences precursor accumulation, suggesting that splicing has been tuned in phages to modulate generation of important proteins. Together, this work expands our understanding of phage-based intein dissemination and evolution and implies that phages provide a context for evolution of splicing-based regulation. IMPORTANCE Inteins are mobile protein splicing elements found in critical genes across all domains of life. Mycobacterial inteins are of particular interest because of their occurrence in pathogenic species, such as Mycobacterium tuberculosis and Mycobacterium leprae , which harbor inteins in important proteins. We have discovered a similarity in activities of intein-containing proteins among mycobacteriophages and their intein-rich actinobacterial hosts, with implications for both posttranslational regulation by inteins and phages participating in horizontal intein transfer. Our demonstration of multiple insertion points within active centers of phage proteins implies independent invasion events, indicating the importance of intein maintenance at specific functional sites. The variable conservation of a catalytic splicing residue, leading to profoundly altered splicing rates, points to the regulatory potential of inteins and to mycobacteriophages playing a role in intein evolution. Collectively, these results suggest inteins as posttranslational regulators and mycobacteriophages as both vehicles for intein distribution and incubators for intein evolution.

scholarly journals Computational identification of miRNAs and their targets from Crocus sativus L.

2012 ◽  
Vol 64 (1) ◽  
pp. 65-70 ◽  
Author(s):  
Praveen Guleria ◽  
Deepmala Goswami ◽  
Kumar Yadav
Keyword(s):  
Posttranslational Modifications ◽  
Stress Responses ◽  
Mrna Export ◽  
Crocus Sativus ◽  
Est Database ◽  
Computational Approaches ◽  
Export Protein ◽  
Identification And Characterization ◽  
Crocus Sativus L

miRNAs are smaller known entities that perform several crucial regulatory roles in plants. Identification and characterization of miRNAs has been a challenging task which has become simplified with the development of computational approaches. In the present study, three novel miRNAs were predicted from Crocus sativus by computational approaches. A total 6767 ESTs were mined from the EST database available at NCBI. The software MicroPC was utilized which resulted with the prediction of three miRNAs, csa-miR1, csa-miR2 and csa-miR3. Targets were predicted for the respective miRNAs using the miRU2 program. The identified targets have a role in plant growth, senescence, disease resistance and various stress responses. In addition, some targets are involved in mRNA export, protein synthesis and posttranslational modifications.

Identification and characterization of AtI-2, an Arabidopsis homologue of an ancient protein phosphatase 1 (PP1) regulatory subunit

2011 ◽  
Vol 435 (1) ◽  
pp. 73-83 ◽  
Author(s):  
George W. Templeton ◽  
Mhairi Nimick ◽  
Nicholas Morrice ◽  
David Campbell ◽  
Marilyn Goudreault ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Biochemical Characterization ◽  
Affinity Purification ◽  
Bioinformatic Analysis ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Identification And Characterization ◽  
Eukaryotic Genomes

PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.

scholarly journals Toxoplasma gondii serine hydrolases regulate parasite lipid mobilization during growth and replication within the host

2020 ◽  
Author(s):  
Ouma Onguka ◽  
Brett M. Babin ◽  
Markus Lakemeyer ◽  
Ian T. Foe ◽  
Neri Amara ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Biochemical Characterization ◽  
Lipid Synthesis ◽  
Protozoan Parasite ◽  
List Type ◽  
Serine Hydrolases ◽  
Metabolizing Enzymes ◽  
Thiol Esters ◽  
Intracellular Protozoan Parasite

SummaryThe intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications.HighlightsBioinformatic and biochemical characterization of T. gondii serine hydrolases reveals substrate preference between enzymes with similar catalytic foldT. gondii serine hydrolases previously thought to be depalmitoylases are lipid metabolizing enzymesT. gondii lipid metabolism pathways utilize enzymes that are viable therapeutic targets

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