Expanding the paradigm of thiol redox in the thermophilic root of life

Heinemann, Joshua; Hamerly, Timothy; Maaty, Walid S.; Movahed, Navid; Steffens, Joseph; Reeves, Benjamin D.; Hilmer, Jonathan K.; Therien, Jesse; Grieco, Paul A.; Peters, John W.; Bothner, Brian

doi:10.1016/j.bbagen.2013.08.009

Cited by 19 publications

(24 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Even if the proteins were prepared from the original organisms, disulfide bonds could be formed through aerobic manipulation of the proteins, and in such cases, no one can claim that the redox states of the proteins are the same as those are in vivo. By using thiol-labeling techniques, a few reports have demonstrated that there are a large number of proteins possessing disulfide bonds in the cells of P. aerophilum and S. solfataricus (6,21); however, as far as we could ascertain, there is no report that has clearly shown the redox state of a certain protein-i.e., what percentage of the protein is in the oxidized form-in the cells of thermophiles, while Heinemann et al recently reported that the cytosol of S. solfataricus lacks reduced small molecule thiols like glutathione and that glutathione is mainly in the oxidized, disulfide-bonded form in the cells (21).…”

Section: Importancementioning

confidence: 71%

“…Because ϳ80°C is reported as the optimal growth temperature of S. solfataricus (50), and because the inactivation of recom- binant DMD occurred at similar temperatures when Cys210 was replaced by serine or when the disulfide bond was reduced, it is tempting to imagine that the formation of the disulfide bond is required for DMD to exhibit activity at the growth temperature of S. solfataricus. However, even though frequent in vivo formation of disulfide bonds in some thermophilic archaea has been proven (6,21), the efficiency of disulfide formation in each kind of protein is still unclear. Thus, we attempted to examine the formation of the disulfide bond of DMD in the cells of S. solfataricus using nonreducing SDS-PAGE and Western blotting.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

et al. 2015

View full text Add to dashboard Cite

In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of ␤-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus. IMPORTANCEThis study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance.

show abstract

Section: Importancementioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

et al. 2015

View full text Add to dashboard Cite

show abstract

“…Detection of metabolites was performed via HPLC separation with ESI-MS (electrospray mass spectrometry) detection in the Montana State University Mass Spectrometry Core Facility [33,34,35]. HPLC was performed with an aqueous normal-phase, hydrophilic interaction chromatography (ANP/HILIC) HPLC column.…”

Section: Methodsmentioning

confidence: 99%

Candidate mediators of chondrocyte mechanotransduction via targeted and untargeted metabolomic measurements

Jutila

Zignego

Hwang

et al. 2014

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

Chondrocyte mechanotransduction is the process by which cartilage cells transduce mechanical loads into biochemical and biological signals. Previous studies have identified several pathways by which chondrocytes transduce mechanical loads, yet a general understanding of which signals are activated and in what order remains elusive. This study was performed to identify candidate mediators of chondrocyte mechanotransduction using SW1353 chondrocytes embedded in physiologically stiff agarose. Dynamic compression was applied to cell-seeded constructs for 0–30 minutes, followed immediately by whole-cell metabolite extraction. Metabolites were detected via LC-MS, and compounds of interest were identified via database searches. We found several metabolites which were statistically different between the experimental groups, and we report the detection of 5 molecules which are not found in metabolite databases of known compounds indicating potential novel molecules. Targeted studies to quantify the response of central energy metabolites to compression found a transient increase in the ratio of NADP+ to NADPH and a continual decrease in the ratio of GDP to GTP, suggesting a flux of energy into the TCA cycle. These data are consistent with the remodeling of cytoskeletal components by mechanically induced signaling, and add substantial new data to a complex picture of how chondrocytes transduce mechanical loads.

show abstract

“…Intracellular metabolites from I. hospitalis and I. hospitalis – N. equitans co-cultures for LC-MS and NMR analysis were extracted using a 50% aqueous (v/v) MeOH extraction, modified from a previously published protocol (Heinemann et al ., 2014). Briefly, frozen cell pellets weighing 50 mg for LC-MS analysis and 115 mg for NMR analysis, were re-suspended in 300 μL of 50% MeOH (v/v), vortexed for 30 seconds, and lysed by sonication for 5 minutes on ice.…”

Section: Methodsmentioning

confidence: 99%

Untargeted metabolomics studies employing NMR and LC–MS reveal metabolic coupling between Nanoarcheum equitans and its archaeal host Ignicoccus hospitalis

et al. 2014

Self Cite

View full text Add to dashboard Cite

Interspecies interactions are the basis of microbial community formation and infectious diseases. Systems biology enables the construction of complex models describing such interactions, leading to a better understanding of disease states and communities. However, before interactions between complex organisms can be understood, metabolic and energetic implications of simpler real-world host-microbe systems must be worked out. To this effect, untargeted metabolomics experiments were conducted and integrated with proteomics data to characterize key molecular-level interactions between two hyperthermophilic microbial species, both of which have reduced genomes. Metabolic changes and transfer of metabolites between the archaea Ignicoccus hospitalis and Nanoarcheum equitans were investigated using integrated LC-MS and NMR metabolomics. The study of such a system is challenging, as no genetic tools are available, growth in the laboratory is challenging, and mechanisms by which they interact are unknown. Together with information about relative enzyme levels obtained from shotgun proteomics, the metabolomics data provided useful insights into metabolic pathways and cellular networks of I. hospitalis that are impacted by the presence of N. equitans, including arginine, isoleucine, and CTP biosynthesis. On the organismal level, the data indicate that N. equitans exploits metabolites generated by I. hospitalis to satisfy its own metabolic needs. This finding is based on N. equitans’s consumption of a significant fraction of the metabolite pool in I. hospitalis that cannot solely be attributed to increased biomass production for N. equitans. Combining LC-MS and NMR metabolomics datasets improved coverage of the metabolome and enhanced the identification and quantitation of cellular metabolites.

show abstract

Expanding the paradigm of thiol redox in the thermophilic root of life

Cited by 19 publications

References 38 publications

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus

Candidate mediators of chondrocyte mechanotransduction via targeted and untargeted metabolomic measurements

Untargeted metabolomics studies employing NMR and LC–MS reveal metabolic coupling between Nanoarcheum equitans and its archaeal host Ignicoccus hospitalis

Contact Info

Product

Resources

About