scholarly journals Translational GTPase BipA Is Involved in the Maturation of a Large Subunit of Bacterial Ribosome at Suboptimal Temperature

2021 ◽  
Vol 12 ◽  
Author(s):  
Kwok Jian Goh ◽  
Rya Ero ◽  
Xin-Fu Yan ◽  
Jung-Eun Park ◽  
Binu Kundukad ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Protein A ◽  
Protein Translation ◽  
Rna Metabolism ◽  
Soft Agar ◽  
Mass Spectrometry Analysis ◽  
Ribosome Assembly ◽  
Spectrometry Analysis ◽  
Bacterial Ribosome

BPI-inducible protein A (BipA), a highly conserved paralog of the well-known translational GTPases LepA and EF-G, has been implicated in bacterial motility, cold shock, stress response, biofilm formation, and virulence. BipA binds to the aminoacyl-(A) site of the bacterial ribosome and establishes contacts with the functionally important regions of both subunits, implying a specific role relevant to the ribosome, such as functioning in ribosome biogenesis and/or conditional protein translation. When cultured at suboptimal temperatures, the Escherichia coli bipA genomic deletion strain (ΔbipA) exhibits defects in growth, swimming motility, and ribosome assembly, which can be complemented by a plasmid-borne bipA supplementation or suppressed by the genomic rluC deletion. Based on the growth curve, soft agar swimming assay, and sucrose gradient sedimentation analysis, mutation of the catalytic residue His78 rendered plasmid-borne bipA unable to complement its deletion phenotypes. Interestingly, truncation of the C-terminal loop of BipA exacerbates the aforementioned phenotypes, demonstrating the involvement of BipA in ribosome assembly or its function. Furthermore, tandem mass tag-mass spectrometry analysis of the ΔbipA strain proteome revealed upregulations of a number of proteins (e.g., DeaD, RNase R, CspA, RpoS, and ObgE) implicated in ribosome biogenesis and RNA metabolism, and these proteins were restored to wild-type levels by plasmid-borne bipA supplementation or the genomic rluC deletion, implying BipA involvement in RNA metabolism and ribosome biogenesis. We have also determined that BipA interacts with ribosome 50S precursor (pre-50S), suggesting its role in 50S maturation and ribosome biogenesis. Taken together, BipA demonstrates the characteristics of a bona fide 50S assembly factor in ribosome biogenesis.

scholarly journals Refining the composition of the Arabidopsis thaliana 80S cytosolic ribosome

2019 ◽  
Author(s):  
Karzan Jalal Salih ◽  
Owen Duncan ◽  
Lei Li ◽  
Josua Troesch ◽  
A. Harvey Millar
Keyword(s):  
Arabidopsis Thaliana ◽  
Ribosome Biogenesis ◽  
Cell Function ◽  
Protein A ◽  
Mass Spectrometry Analysis ◽  
Turnover Rates ◽  
80S Ribosome ◽  
Spectrometry Analysis ◽  
Peptide Mass

AbstractThe cytosolic 80S ribosome is composed of protein and RNA molecules and its function in protein synthesis is modulated through interaction with other cytosolic components. Defining the role of each of the proteins associated with ribosomes in plants is a major challenge which is hampered by difficulties in attribution of different proteins to roles in ribosome biogenesis, the mature cytosolic ribosome (r-proteins) or to the broader translatome associated with functioning ribosomes. Here we refined the core r-protein composition in plants by determining the abundance of proteins in low, partially and highly purified ribosomal samples from Arabidopsis thaliana cell cultures. To characterise this list of proteins further we determined their degradation (KD) and synthesis (KS) rate by progressive labelling with 15N combined with peptide mass spectrometry analysis. The turnover rates of 55 r-proteins, including 26 r-proteins from the 40S subunit and 29 r-proteins from the 60S subunit could be determined. Overall, ribosome proteins showed very similar KD and KS rates suggesting that half of the ribosome population is replaced every 3-4 days. Three proteins showed significantly shorter half-lives; ribosomal protein P0D (RPP0D) with a half-life of 0.5 days and RACK1b and c with half-lives of 1-1.4 days. The ribosomal RPP0D protein is a homolog of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role as a 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins in Arabidopsis. Of this set, 26 were enriched more than 10-fold during ribosome purification. A number have known roles in translation or ribosome-association while others are newly proposed ribosome-associated factors in plants. This analysis provides a more robust understanding of Arabidopsis ribosome content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and reveals how protein turnover can identify r-proteins involved in ribosome biogenesis or regulation in plants. Data are available via ProteomeXchange with identifier PXD012839.

scholarly journals Re-Discovery of Giardiavirus: Genomic and Functional Analysis of Viruses from Giardia duodenalis Isolates

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 654
Author(s):  
Gianluca Marucci ◽  
Ilaria Zullino ◽  
Lucia Bertuccini ◽  
Serena Camerini ◽  
Serena Cecchetti ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Diarrheal Disease ◽  
Current Knowledge ◽  
Biological Significance ◽  
Giardia Duodenalis ◽  
Protozoan Parasite ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Animal Origin ◽  
Spectrometry Analysis

Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/−2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.

scholarly journals Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti

2019 ◽  
Vol 116 (38) ◽  
pp. 19136-19144 ◽  
Author(s):  
Giel P. Göertz ◽  
Joyce W. M. van Bree ◽  
Anwar Hiralal ◽  
Bas M. Fernhout ◽  
Carmen Steffens ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Zika Virus ◽  
Affinity Purification ◽  
Mechanistic Model ◽  
Virus Transmission ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Small Interfering Rnas ◽  
Spectrometry Analysis ◽  
Mosquito Infection

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

scholarly journals Evidence for Direct Protein-Protein Interaction between Members of the Enterobacterial Hha/YmoA and H-NS Families of Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 629-635 ◽  
Author(s):  
J. M. Nieto ◽  
C. Madrid ◽  
E. Miquelay ◽  
J. L. Parra ◽  
S. Rodríguez ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein A ◽  
Nitrilotriacetic Acid ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Protein Protein Interaction ◽  
New Family ◽  
Missense Mutant ◽  
First Time

ABSTRACT Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin α-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni2+-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha′ proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.

scholarly journals The Impact of the Stringent Response on TRAFAC GTPases and Prokaryotic Ribosome Assembly

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1313 ◽  
Author(s):  
Bennison ◽  
Irving ◽  
Corrigan
Keyword(s):  
Ribosomal Proteins ◽  
Cellular Response ◽  
Ribosome Biogenesis ◽  
Stringent Response ◽  
Protein Translation ◽  
Bound State ◽  
Ribosome Assembly ◽  
Rrna Transcription ◽  
Stress Sensing ◽  
The Impact

Many facets of ribosome biogenesis and function, including ribosomal RNA (rRNA) transcription, 70S assembly and protein translation, are negatively impacted upon induction of a nutrient stress-sensing signalling pathway termed the stringent response. This stress response is mediated by the alarmones guanosine tetra- and penta-phosphate ((p)ppGpp), the accumulation of which leads to a massive cellular response that slows growth and aids survival. The 70S bacterial ribosome is an intricate structure, with assembly both complex and highly modular. Presiding over the assembly process is a group of P-loop GTPases within the TRAFAC (Translation Factor Association) superclass that are crucial for correct positioning of both early and late stage ribosomal proteins (r-proteins) onto the rRNA. Often described as ‘molecular switches’, members of this GTPase superfamily readily bind and hydrolyse GTP to GDP in a cyclic manner that alters the propensity of the GTPase to carry out a function. TRAFAC GTPases are considered to act as checkpoints to ribosome assembly, involved in binding to immature sections in the GTP-bound state, preventing further r-protein association until maturation is complete. Here we review our current understanding of the impact of the stringent response and (p)ppGpp production on ribosome maturation in prokaryotic cells, focusing on the inhibition of (p)ppGpp on GTPase-mediated subunit assembly, but also touching upon the inhibition of rRNA transcription and protein translation.

scholarly journals Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

2015 ◽  
Vol 81 (24) ◽  
pp. 8330-8338 ◽  
Author(s):  
Hao Yu ◽  
Hongzhi Tang ◽  
Yangyang Li ◽  
Ping Xu
Keyword(s):  
Inductively Coupled Plasma ◽  
Large Subunit ◽  
Sole Source ◽  
Small Subunit ◽  
Binding Domain ◽  
Mass Spectrometry Analysis ◽  
Resting Cells ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Nicotine Degradation

ABSTRACTOchrobactrumsp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppASandvppAL[subunits S and L, respectively]). ThevppAgenes were heterologously expressed in the non-nicotine-degrading strainsEscherichia coliDH5α andPseudomonas putidaKT2440; only thePseudomonasstrain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.

Linking Defective Ribosome Maturation to Shwachman-Diamond Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-36-SCI-36
Author(s):  
Alan John Warren
Keyword(s):  
Ribosome Biogenesis ◽  
Conflicts Of Interest ◽  
Ribosomal Subunit ◽  
Protein Translation ◽  
Ribosome Assembly ◽  
Genetic Dissection ◽  
Gtp Hydrolysis ◽  
Shwachman Diamond Syndrome ◽  
Novel Therapeutic Strategies ◽  
Syndrome Protein

Abstract Ribosomes are RNA-protein machines that translate the genetic information encoded by the mRNA template in all living cells. Recent high-resolution structures of the ribosome have revolutionized our understanding of protein translation. However, the mechanisms of ribosome assembly and the surveillance mechanisms that monitor this process and couple it to growth are poorly understood. Causative mutations and deletions of genes involved in ribosome biogenesis define an emerging group of disorders known as the ribosomopathies. Recent work from my laboratory strongly supports the hypothesis that Shwachman-Diamond syndrome (SDS) is a ribosomopathy caused by defective maturation of the large ribosomal subunit. Elucidation of the specific function of the SBDS protein that is deficient in SDS is revealing unexpected new insights that extend our understanding of the mechanisms underlying the late cytoplasmic steps of ribosome assembly and the quality control surveillance pathways that monitor 60S maturation. Genetic dissection of this pathway may inform novel therapeutic strategies for SDS. 1. Wong C.C., Traynor D., Basse N., Kay R.R., Warren A.J. Defective ribosome assembly in Shwachman-Diamond syndrome. Plenary Paper, Blood. 2011 Oct 20;118(16):4305-12. 2. Finch A.J., Hilcenko C., Basse N., Drynan L.F., Goyenechea B., Menne T.F., González Fernández Á., Simpson P., D’Santos C.S., Arends M.J., Donadieu J., Bellanné-Chantelot C., Costanzo M., Boone C., McKenzie A.N., Freund S.M., Warren A.J. Uncoupling of GTP hydrolysis from eIF6 release on the ribosome causes Shwachman-Diamond syndrome. Genes and Development (2011) 25: 917-929. 3. Menne T.M., Goyenechea B., Sánchez-Puig N., Wong C.C., Tonkin L.M., Ancliff P., Brost R.L., Costanzo M., Boone C. and Warren A.J. The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nature Genetics (2007) 39: 486-95. Disclosures: No relevant conflicts of interest to declare.

Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages

2000 ◽  
Vol 278 (5) ◽  
pp. L1025-L1031 ◽  
Author(s):  
Sha Zhu ◽  
Khaled F. Basiouny ◽  
John P. Crow ◽  
Sadis Matalon
Keyword(s):  
Nitric Oxide ◽  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Dose Dependent Fashion ◽  
Carbonyl Formation ◽  
Oxide Synthase ◽  
Dose Dependent

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO2. In the presence of CO2, lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion ofl-[U-14C]arginine tol-[U-14C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 ± 3 vs. 6 ± 1 (SE) nmol ⋅ 6.5 h−1 ⋅ 106AMs−1]. Western blotting studies of immunoprecipitated SP-A indicated that CO2 enhanced SP-A nitration by AMs and decreased carbonyl formation. CO2(0–1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO2. Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr164 and Tyr166) in the absence of CO2 and three tyrosines (Tyr164, Tyr166, and Tyr161) in the presence of 1.2 mM CO2. These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO2 increased nitration, at least partially, by enhancing enzymatic nitric oxide production.

scholarly journals Evaluation of a Vaccine Formulation against Streptococcus pneumoniae Based on Choline-Binding Proteins

2014 ◽  
Vol 22 (2) ◽  
pp. 213-220 ◽  
Author(s):  
Eliane N. Miyaji ◽  
Cintia F. M. Vadesilho ◽  
Maria Leonor S. Oliveira ◽  
André Zelanis ◽  
David E. Briles ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Proteins ◽  
Choline Chloride ◽  
Protein A ◽  
Surface Protein ◽  
Mass Spectrometry Analysis ◽  
Effective Vaccine ◽  
Lethal Challenge ◽  
Content Type ◽  
Spectrometry Analysis

ABSTRACTStreptococcus pneumoniaehas proteins that are attached to its surface by binding to phosphorylcholine of teichoic and lipoteichoic acids. These proteins are known as choline-binding proteins (CBPs). CBPs are an interesting alternative for the development of a cost-effective vaccine, and PspA (pneumococcal surface protein A) is believed to be the most important protective component among the different CBPs. We sought to use CBPs eluted from pneumococci as an experimental vaccine. Since PspA shows variability between isolates, we constructed strains producing different PspAs. We used the nonencapsulated Rx1 strain, which produces PspA from clade 2 (PspA2), to generate apspA-knockout strain (Rx1 ΔpspA) and strains expressing PspA from clade 1 (Rx1pspA1) and clade 4 (Rx1pspA4). We grew Rx1, Rx1 ΔpspA, Rx1pspA1, and Rx1pspA4in Todd-Hewitt medium containing 0.5% yeast extract and washed cells in 2% choline chloride (CC). SDS-PAGE analysis of the proteins recovered by a CC wash showed few bands, and the CBPs PspA and PspC (pneumococcal surface protein C) were identified by mass spectrometry analysis. Subcutaneous immunization of mice with these full-length native proteins without adjuvant led to significantly higher rates of survival than immunization with diluent after an intranasal lethal challenge with two pneumococcal strains and also after a colonization challenge with one strain. Importantly, immunization with recombinant PspA4 (rPspA4) without adjuvant did not elicit significant protection.

scholarly journals Discovery of a small molecule that inhibits bacterial ribosome biogenesis

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jonathan M Stokes ◽  
Joseph H Davis ◽  
Chand S Mangat ◽  
James R Williamson ◽  
Eric D Brown
Keyword(s):  
Small Molecule ◽  
Ribosome Biogenesis ◽  
Anticonvulsant Drug ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Bacterial Ribosome ◽  
Cold Sensitive

While small molecule inhibitors of the bacterial ribosome have been instrumental in understanding protein translation, no such probes exist to study ribosome biogenesis. We screened a diverse chemical collection that included previously approved drugs for compounds that induced cold sensitive growth inhibition in the model bacterium Escherichia coli. Among the most cold sensitive was lamotrigine, an anticonvulsant drug. Lamotrigine treatment resulted in the rapid accumulation of immature 30S and 50S ribosomal subunits at 15°C. Importantly, this was not the result of translation inhibition, as lamotrigine was incapable of perturbing protein synthesis in vivo or in vitro. Spontaneous suppressor mutations blocking lamotrigine activity mapped solely to the poorly characterized domain II of translation initiation factor IF2 and prevented the binding of lamotrigine to IF2 in vitro. This work establishes lamotrigine as a widely available chemical probe of bacterial ribosome biogenesis and suggests a role for E. coli IF2 in ribosome assembly.

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