scholarly journals Taking a Step Back from Back-Translocation: an Integrative View of LepA/EF4's Cellular Function

2017 ◽  
Vol 37 (12) ◽  
Author(s):  
Jalyce L. E. Heller ◽  
Rajashekhar Kamalampeta ◽  
Hans-Joachim Wieden
Keyword(s):  
Protein Synthesis ◽  
Functional Role ◽  
Ribosome Biogenesis ◽  
Cellular Function ◽  
Specific Function ◽  
Ribosome Stalling ◽  
Terminal Domain ◽  
Integrative View ◽  
Domain Arrangement

ABSTRACT Protein synthesis, the translation of mRNA into a polypeptide facilitated by the ribosome, is assisted by a variety of protein factors, some of which are GTPases. In addition to four highly conserved and well-understood GTPases with known function, there are also a number of noncanonical GTPases that are implicated in translation but whose functions are not fully understood. LepA/EF4 is one of these noncanonical GTPases. It is highly conserved and present in bacteria, mitochondria, and chloroplasts, but its functional role in the cell remains unknown. LepA's sequence and domain arrangement are very similar to those of other translational GTPases, but it contains a unique C-terminal domain (CTD) that is likely essential to its specific function in the cell. Three main hypotheses about the function of LepA have been brought forward to date: (i) LepA is a back-translocase, (ii) LepA relieves ribosome stalling or facilitates sequestration, and (iii) LepA is involved in ribosome biogenesis. This review examines the structural and biochemical information available on bacterial LepA and discusses it on the background of the available in vivo information from higher organisms in order to broaden the view regarding LepA's functional role in the cell and how the structure of its unique CTD might be involved in facilitating this role.

scholarly journals Regulation of ribosomal protein synthesis in mycobacteria: autogenous control of rpsO

2021 ◽  
Author(s):  
Leonid V Aseev ◽  
Ludmila S Koledinskaya ◽  
Oksana S Bychenko ◽  
Irina V Boni
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribosome Biogenesis ◽  
Bacterial Species ◽  
Shuttle Vector ◽  
Gc Content ◽  
E Coli ◽  
Autogenous Control ◽  
Autogenous Regulation

ABSTRACTAutogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. Our main goal was to develop techniques for investigating the r-protein synthesis regulation in mycobacteria, Gram-positive organisms with a high GC-content, which has never been addressed. We started with the rpsO gene known to be autoregulated by its product, r-protein S15, in a broad range of bacterial species. To study the in vivo regulation of rpsO from Mycobacterium smegmatis (Msm), we first applied an approach based on chromosomally integrated Msm rpsO’-’lacZ reporters by using E. coli as a surrogate host. The β-galactosidase assay has shown that mycobacterial rpsO expression is feedback regulated at the translation level in the presence of Msm S15 in trans, like in E. coli. Next, to overcome difficulties caused by the inefficiency of mycobacterial gene expression in E. coli, we created a fluorescent reporter system based on M. smegmatis. To this end, the integrative shuttle plasmid pMV306 was modified to provide insertion of the Msm or Mtb (M. tuberculosis) rpsO-egfp reporters into the Msm chromosome, and a novel E. coli-mycobacteria replicative shuttle vector, pAMYC, a derivative of pACYC184, was built. Analysis of the eGFP expression in the presence of the pAMYC derivative expressing Msm rpsO vs an empty vector confirms the autogenous regulation of the rpsO gene in mycobacteria. Additionally, we have revealed that the mycobacterial rpsO core promoters are rather weak and require upstream activating elements to enhance their strength.IMPORTANCEBacterial ribosomes are targets for a majority of as-yet reported antibiotics, hence ribosome biogenesis and its regulation are central for development of new antimicrobials. One of the key mechanisms regulating ribosome biogenesis in bacteria is the autogenous control of r-protein synthesis, which has been so far explored for E. coli and Bacillus spp. but not yet for mycobacteria. Here, we describe experimental approaches for in vivo analysis of mechanisms regulating r-protein synthesis in mycobacteria, including M. tuberculosis, and show, for the first time, that the autogenous control at the translation level is really functioning in these microorganisms. The developed system paves the way for studying various regulatory circuits involving proteins or sRNAs as mRNA- targeting trans-regulators in mycobacteria as well as in other actinobacterial species.

scholarly journals Developmental stage-specific changes in protein synthesis differentially sensitize hematopoietic stem cells and erythroid progenitors to impaired ribosome biogenesis

2020 ◽  
Author(s):  
Jeffrey A. Magee ◽  
Robert A.J. Signer
Keyword(s):  
Stem Cells ◽  
Protein Synthesis ◽  
Ribosome Biogenesis ◽  
Erythroid Differentiation ◽  
Cell Type ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Cell Type Specific

AbstractRibosomopathies encompass a collection of human genetic disorders that often arise from mutations in ribosomal proteins or ribosome biogenesis factors. Despite ubiquitous requirement of ribosomes for protein synthesis, ribosomopathies present with tissue- and cell-type-specific disorders, and blood is particularly affected. Several ribosomopathies present with congenital anemias and bone marrow failure, and accordingly, erythroid lineage cells and hematopoietic stem cells (HSCs) are preferentially impaired by ribosomal dysfunction. However, the factors that influence this cell-type-specific sensitivity are incompletely understood. Here, we show that protein synthesis rates change during HSC and erythroid progenitor ontogeny. Fetal HSCs exhibit significantly higher protein synthesis than adult HSCs. Despite protein synthesis differences, reconstituting activity of both fetal and adult HSCs is severely disrupted by a ribosomal mutation (Rpl24Bst/+). In contrast, fetal erythroid lineage progenitors exhibit significantly lower protein synthesis than their adult counterparts. Protein synthesis declines during erythroid differentiation, but the decline starts earlier in fetal differentiation than in adults. Strikingly, the Rpl24Bst/+ mutation impairs fetal, but not adult erythropoiesis, by impairing proliferation at fetal erythroid progenitor stages with the lowest protein synthesis relative to their adult counterparts. Thus, developmental and cell-type-specific changes in protein synthesis can sensitize hematopoietic cells to impaired ribosome biogenesis.Key PointsFetal HSCs synthesize much more protein per hour than young adult HSCs in vivoFetal erythroid progenitors synthesize much less protein per hour than young adult erythroid progenitors in vivoDifferences in protein synthesis dynamics distinguish fetal and adult erythroid differentiationA ribosomal mutation that reduces protein synthesis impairs fetal and adult HSCsReduced protein synthesis impairs fetal but not adult erythroid progenitors

Respiratory energy requirements and rate of protein turnover in vivo determined by the use of an inhibitor of protein synthesis and a probe to assess its effect

1994 ◽  
Vol 92 (4) ◽  
pp. 585-594 ◽  
Author(s):  
T. J. Bouma ◽  
R. De Visser ◽  
J. H. J. A. Janssen ◽  
M. J. De Kock ◽  
P H. Van Leeuwen ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Energy Requirements ◽  
Inhibitor Of Protein Synthesis

Rapid in vivo induction of leukocyte tissue factor mRNA and protein synthesis following low dose endotoxin administration to rabbits

2001 ◽  
Vol 2 (3) ◽  
pp. 188-195 ◽  
Author(s):  
Tara C Brutzki ◽  
Myron J Kulczycky ◽  
Leslie Bardossy ◽  
Bryan J Clarke ◽  
Morris A Blajchman
Keyword(s):  
Protein Synthesis ◽  
Tissue Factor ◽  
Low Dose ◽  
Endotoxin Administration ◽  
In Vivo Induction

scholarly journals The C-terminal domain of feline and bovine SAMHD1 proteins has a crucial role in lentiviral restriction

2020 ◽  
Vol 295 (13) ◽  
pp. 4252-4264 ◽  
Author(s):  
Chu Wang ◽  
Kaikai Zhang ◽  
Lina Meng ◽  
Xin Zhang ◽  
Yanan Song ◽  
...  
Keyword(s):  
Proteasomal Degradation ◽  
Acid Sites ◽  
Accessory Protein ◽  
Restriction Profile ◽  
Biological Functions ◽  
Terminal Domain ◽  
Functional Regions ◽  
Viral Restriction ◽  
Primate Lentiviruses

SAM and HD domain-containing protein 1 (SAMHD1) is a host factor that restricts reverse transcription of lentiviruses such as HIV in myeloid cells and resting T cells through its dNTP triphosphohydrolase (dNTPase) activity. Lentiviruses counteract this restriction by expressing the accessory protein Vpx or Vpr, which targets SAMHD1 for proteasomal degradation. SAMHD1 is conserved among mammals, and the feline and bovine SAMHD1 proteins (fSAM and bSAM) restrict lentiviruses by reducing cellular dNTP concentrations. However, the functional regions of fSAM and bSAM that are required for their biological functions are not well-characterized. Here, to establish alternative models to investigate SAMHD1 in vivo, we studied the restriction profile of fSAM and bSAM against different primate lentiviruses. We found that both fSAM and bSAM strongly restrict primate lentiviruses and that Vpx induces the proteasomal degradation of both fSAM and bSAM. Further investigation identified one and five amino acid sites in the C-terminal domain (CTD) of fSAM and bSAM, respectively, that are required for Vpx-mediated degradation. We also found that the CTD of bSAM is directly involved in mediating bSAM's antiviral activity by regulating dNTPase activity, whereas the CTD of fSAM is not. Our results suggest that the CTDs of fSAM and bSAM have important roles in their antiviral functions. These findings advance our understanding of the mechanism of fSAM- and bSAM-mediated viral restriction and might inform strategies for improving HIV animal models.

scholarly journals The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor

2021 ◽  
Vol 22 (11) ◽  
pp. 5871
Author(s):  
Almerinda Di Venere ◽  
Eleonora Nicolai ◽  
Velia Minicozzi ◽  
Anna Maria Caccuri ◽  
Luisa Di Paola ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Strong Dependence ◽  
Conformational Dynamics ◽  
Receptor Interaction ◽  
Tnf Receptor ◽  
Oligomeric State ◽  
Dynamic Simulations ◽  
Associated Factor ◽  
Terminal Domain

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.

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