scholarly journals HIV-1 hypermethylated guanosine cap licenses specialized translation unaffected by mTOR

2021 ◽  
Vol 119 (1) ◽  
pp. e2105153118
Author(s):  
Gatikrushna Singh ◽  
Bradley Seufzer ◽  
Zhenwei Song ◽  
Dora Zucko ◽  
Xiao Heng ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Immune Cells ◽  
Messenger Rna ◽  
De Novo ◽  
Protein Translation ◽  
Primer Binding Site ◽  
Virion Protein ◽  
Down Regulation ◽  
Mtor Inhibition ◽  
Hiv 1

Appended to the 5′ end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1–reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5′ untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.

scholarly journals SAMD9L autoinflammatory or ataxia pancytopenia disease mutations activate cell-autonomous translational repression

2021 ◽  
Vol 118 (34) ◽  
pp. e2110190118
Author(s):  
Amanda J. Russell ◽  
Paul E. Gray ◽  
John B. Ziegler ◽  
Yae Jean Kim ◽  
Sandy Smith ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Autoinflammatory Disease ◽  
Fluorescent Protein ◽  
Single Cell Analysis ◽  
De Novo ◽  
Protein Translation ◽  
Translational Repression ◽  
Translational Repressor ◽  
Activate Cell ◽  
Green Fluorescent

Sterile α motif domain-containing protein 9-like (SAMD9L) is encoded by a hallmark interferon-induced gene with a role in controlling virus replication that is not well understood. Here, we analyze SAMD9L function from the perspective of human mutations causing neonatal-onset severe autoinflammatory disease. Whole-genome sequencing of two children with leukocytoclastic panniculitis, basal ganglia calcifications, raised blood inflammatory markers, neutrophilia, anemia, thrombocytopaenia, and almost no B cells revealed heterozygous de novo SAMD9L mutations, p.Asn885Thrfs*6 and p.Lys878Serfs*13. These frameshift mutations truncate the SAMD9L protein within a domain a region of homology to the nucleotide-binding and oligomerization domain (NOD) of APAF1, ∼80 amino acids C-terminal to the Walker B motif. Single-cell analysis of human cells expressing green fluorescent protein (GFP)-SAMD9L fusion proteins revealed that enforced expression of wild-type SAMD9L repressed translation of red fluorescent protein messenger RNA and globally repressed endogenous protein translation, cell autonomously and in proportion to the level of GFP-SAMD9L in each cell. The children’s truncating mutations dramatically exaggerated translational repression even at low levels of GFP-SAMD9L per cell, as did a missense Arg986Cys mutation reported recurrently as causing ataxia pancytopenia syndrome. Autoinflammatory disease associated with SAMD9L truncating mutations appears to result from an interferon-induced translational repressor whose activity goes unchecked by the loss of C-terminal domains that may normally sense virus infection.

scholarly journals Microglia perform local protein synthesis at perisynaptic and phagocytic structures

2021 ◽  
Author(s):  
Michael J. Vasek ◽  
Jelani D. Deajon-Jackson ◽  
Yating Liu ◽  
Haley W. Crosby ◽  
Jiwon Yi ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
De Novo ◽  
Protein Translation ◽  
Local Translation ◽  
Slice Preparation ◽  
Local Protein Synthesis ◽  
Phagocytic Cups ◽  
Regulate Protein ◽  
Lysosomal Proteins ◽  
Local Protein

AbstractRecent studies have illuminated the importance of several key signaling pathways in regulating the dynamic surveillance and phagocytic activity of microglia. Yet little is known about how these signals result in the assembly of phagolysosomal machinery near targets of phagocytosis, especially in processes distal from the microglial soma. Neurons, astrocytes, and oligodendrocytes locally regulate protein translation within distal processes. Therefore, we tested whether there is regulated local translation within peripheral microglia processes (PeMPs). We show that PeMPs contain ribosomes which engage in de novo protein synthesis, and these associate with a subpool of transcripts involved in pathogen defense, motility, and phagocytosis. Using a live slice preparation, we further show that acute translation blockade impairs the formation of PeMP phagocytic cups, the localization of lysosomal proteins within them, and phagocytosis. Collectively, these data argue for a regulated local translation in PeMPs and indicate a need for new translation to support dynamic microglial function.

scholarly journals Functional Characterization of the Canine Heme-Regulated eIF2αKinase: Regulation of Protein Synthesis

2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Kimon C. Kanelakis ◽  
Jayashree Pyati ◽  
Pamela C. Wagaman ◽  
Jui Chang Chuang ◽  
Young Yang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Expression System ◽  
Functional Characterization ◽  
Protein Translation ◽  
In Vitro Translation ◽  
Initiation Factor ◽  
Suitable Model ◽  
Dependent Manner

The heme-regulated inhibitor (HRI) negatively regulates protein synthesis by phosphorylating eukaryotic initiation factor-2α(eIF2α) thereby inhibiting protein translation. The importance of HRI in regulating hemoglobin synthesis in erythroid cells makes it an attractive molecular target in need of further characterization. In this work, we have cloned and expressed the canine form of the HRI kinase. The canine nucleotide sequence has 86%, 82%, and 81% identity to the human, mouse, and rat HRI, respectively. It was noted that an isoleucine residue in the ATP binding site of human, rat, and mouse HRI is replaced by a valine in the canine kinase. The expression of canine HRI protein by in vitro translation using wheat germ lysate or in Sf9 cells using a baculovirus expression system was increased by the addition of hemin. Following purification, the canine protein was found to be 72 kD and showed kinase activity determined by its ability to phosphorylate a synthetic peptide substrate. Quercetin, a kinase inhibitor known to inhibit mouse and human HRI, inhibits canine HRI in a concentration-dependent manner. Additionally, quercetin is able to increase de novo protein synthesis in canine reticulocytes. We conclude that the canine is a suitable model species for studying the role of HRI in erythropoiesis.

ON THE MECHANISM OF HORMONE ACTION. VII.

1968 ◽  
Vol 57 (1) ◽  
pp. 33-44 ◽  
Author(s):  
N. Lang ◽  
P. Herrlich ◽  
C. E. Sekeris
Keyword(s):  
Protein Synthesis ◽  
Enzyme Activity ◽  
Messenger Rna ◽  
De Novo ◽  
Hormone Action ◽  
Transaminase Activity ◽  
Active Protein ◽  
Tyrosine Transaminase ◽  
Mechanism Of Hormone Action

ABSTRACT A messenger RNA fraction has been isolated from the liver of cortisol treated rats which when added to an in vitro protein synthesizing sytem gives rise to tyrosine-α-ketoglutarate transaminase activity. Development of enzyme activity is dependent on active protein synthesis and is inhibited by puromycin and erythromycin. It is concluded that the messenger RNA fraction contains the information for the formation of tyrosine transaminase and that the enzyme is formed in vitro by de novo synthesis.

scholarly journals CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation

2015 ◽  
Vol 112 (19) ◽  
pp. 5875-5882 ◽  
Author(s):  
Masahiro Shuda ◽  
Celestino Velásquez ◽  
Erdong Cheng ◽  
Daniel G. Cordek ◽  
Hyun Jin Kwun ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
E3 Ligase ◽  
Protein Translation ◽  
Mitotic Cell ◽  
Cyclin B1 ◽  
Initiation Factor ◽  
Mtor Inhibition ◽  
Viral Oncoprotein ◽  
Interphase Cells ◽  
Dependent Protein

Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m7GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3S10+ mitotic cell population having higher inactive p4E-BP1T37/T46+ saturation levels than pH3S10– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.

scholarly journals Epitope targeting and viral inoculum are determinants of Nef-mediated immune evasion of HIV-1 from cytotoxic T lymphocytes

Blood ◽  
2012 ◽  
Vol 120 (1) ◽  
pp. 100-111 ◽  
Author(s):  
Diana Y. Chen ◽  
Arumugam Balamurugan ◽  
Hwee L. Ng ◽  
William G. Cumberland ◽  
Otto O. Yang
Keyword(s):  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
De Novo ◽  
Down Regulation ◽  
Specific Ctls ◽  
Cell Clearance ◽  
Infected Cells ◽  
The Impact ◽  
Hiv 1

AbstractThe impact of HIV-1 Nef-mediated HLA-I down-regulation on CD8+ cytotoxic T lymphocytes (CTLs) varies by epitope, but the determining factors have not been elucidated. In the present study, we investigated the impact of Nef on the antiviral efficiency of HIV-1–specific CTLs targeting 17 different epitopes to define properties that determine susceptibility to Nef. The impact of Nef was not correlated with the presenting HLA-I type or functional avidity of CTLs, but instead was related directly to the kinetics of infected cell clearance. Whereas Gag-specific CTLs generally were less susceptible to Nef than those targeting other proteins, this was determined by the ability to eliminate infected cells before de novo synthesis of viral proteins, which was also observed for CTLs targeting a Nef epitope. This very early clearance of infected cells depended on virus inoculum, and the required inoculum varied by epitope. These results suggest that whereas Gag-specific CTLs are more likely to recognize infected cells before Nef-mediated HLA-I down-regulation, this varies depending on the specific epitope and virus inoculum. Reduced susceptibility to Nef therefore may contribute to the overall association of Gag-specific CTL responses to better immune control if a sufficient multiplicity of infection is attained in vivo, but this property is not unique to Gag.

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