A newly devised flow cytometric antibody binding assay helps evaluation of dithiothreitol treatment for the inactivation of CD38 on red blood cells.

BACKGROUND AND OBJECTIVES: Anti-CD38 monoclonal antibodies, including daratumumab and isatuximab, often interfere with pretransfusion testing. Dithiothreitol (DTT) treatment of red blood cells (RBCs) negates this interference. However, the optimum DTT concentration and treatment time have not been well defined. Here, we quantified CD38 on RBCs before and after DTT treatment using a flow cytometric antibody binding assay (FABA) to specify the optimum conditions for CD38 inactivation. MATERIALS AND METHODS: For FABA, untreated or DTT-treated RBCs were incubated with fluorescein isothiocyanate-labelled anti-CD38 antibody, in the presence or absence of 100-fold or more excess of unlabelled anti-CD38 antibody, and then analysed by flow cytometry (FCM). Dissociation of CD38-positive and control histograms was determined from the D-value using the Kolmogorov-Smirnov test. The results from FABA were compared with those from conventional FCM, indirect antiglobulin test (IAT) and Western blotting. RESULTS: The results from FABA were more consistent than those from conventional FCM. The D-value was found to be reliable in the analysis of difference between CD38 before and after DTT treatment. Our data showed that 0·0075 mol/l DTT for 30 min is sufficient to inactivate CD38 on RBCs. These results were stable and consistent with the findings from IAT. CONCLUSION: Flow cytometric antibody binding assay is an objective way of evaluating the efficacy of DTT treatment for CD38 on RBCs. This approach allows the detection of a small number of cell surface antigens and will be useful for assessing the various chemical treatments to denature RBC antigens.

Experimental evidence for the thermophilicity of ancestral life.

Theoretical studies have focused on the environmental temperature of the universal common ancestor of life with conflicting conclusions. Here we provide experimental support for the existence of a thermophilic universal common ancestor. We present the thermal stabilities and catalytic efficiencies of nucleoside diphosphate kinases (NDK), designed using the information contained in predictive phylogenetic trees, that seem to represent the last common ancestors of Archaea and of Bacteria. These enzymes display extreme thermal stabilities, suggesting thermophilic ancestries for Archaea and Bacteria. The results are robust to the uncertainties associated with the sequence predictions and to the tree topologies used to infer the ancestral sequences. Moreover, mutagenesis experiments suggest that the universal ancestor also possessed a very thermostable NDK. Because, as we show, the stability of an NDK is directly related to the environmental temperature of its host organism, our results indicate that the last common ancestor of extant life was a thermophile that flourished at a very high temperature.

Profiling of lipids in Thermus thermophilus HB8 grown under various conditions.

The membrane lipids of Thermus species have unique structures. Only four polar lipid species have so far been identified in Thermus thermophilus HB8; namely, are two phosphoglycolipids and two glycolipids, both of which have three branched fatty acid chains. Other lipid molecules may be present; however, they have not been identified so far. To clarify the whole lipid profile of T. thermophilus HB8, we cultured this organism under four different growth (temperature and/or nutrition) conditions and analyzed the compositions of polar lipids and fatty acids by high-performance thin-layer chromatography (HPTLC) and gas chromatograph-mass spectrometry (GCï½°MS), respectively. Thirty-one lipid spots were detected on HPTLC plates and profiled in terms of the presence or absence of phosphate, amino, and sugar groups. Then, we allocated ID numbers to all the spots. Comparative analyses of these polar lipids showed that the diversity of lipid molecules increased under high temperature and minimal medium conditions. In particular, aminolipid species increased under high temperature conditions. As for the fatty acid comparison by GC-MS, iso-branched even-numbered carbon atoms, which are unusual in this organism, significantly increased under the minimal medium condition, suggesting that kinds of branched amino acids at the fatty acid terminus varies under different nutrition conditions. In this study, several unidentified lipids were detected, and elucidation of the lipid structures will provide important information on the environmental adaptation of bacteria.

Crystal structure of adenylosuccinate lyase from the thermophilic bacterium Thermus thermophilus HB8.

Adenylosuccinate lyase (PurB) catalyzes two distinct reactions in the purine nucleotide biosynthetic pathway using the same active site. The ability to recognize two different sets of substrates is of structural and evolutionary interest. In the present study, the crystal structure of PurB from the thermophilic bacterium Thermus thermophilus HB8 (TtPurB) was determined at a resolution of 2.38 Šby molecular replacement using a structure predicted by AlphaFold2 as a template. The asymmetric unit of the TtPurB crystal contained two TtPurB molecules, and some regions were disordered in the crystal structure. The disordered regions were the substrate-binding site and domain 3. TtPurB forms a homotetramer and the monomer is composed of three domains (domains 1, 2 and 3), which is a typical structure for the aspartase/fumarase superfamily. Molecular dynamics simulations with and without substrate/product were performed using a full-length model of TtPurB which was obtained before deletion of the disordered regions. The substrates and products were bound to the model structures during the MD simulations. The fluctuations of amino-acid residues were greater in the disordered regions and became smaller upon the binding of substrate or product. These results demonstrate that the full-length model obtained using AlphaFold2 can be used to generate the coordinates of disordered regions within the crystal structure.

Mechanism for regulation of the putrescine utilization pathway by the transcription factor PuuR in Escherichia coli K-12.

In Escherichia coli, putrescine is metabolized to succinate for use as a carbon and nitrogen source by the putrescine utilization pathway (Puu pathway). One gene in the puu gene cluster encodes a transcription factor, PuuR, which has a helix-turn-helix DNA-binding motif. DNA microarray analysis of an E. coli puuR mutant, in which three amino acid residues in the helix-turn-helix DNA binding motif of PuuR were mutated to alanine to eliminate DNA binding of PuuR, suggested that PuuR is a negative regulator of puu genes. Results of gel shift and DNase I footprint analyses suggested that PuuR binds to the promoter regions of puuA and puuD. The binding of wild-type PuuR to a DNA probe containing PuuR recognition sites was diminished with increasing putrescine concentrations in vitro. These results suggest that PuuR regulates the intracellular putrescine concentration by the transcriptional regulation of genes in the Puu pathway, including puuR itself. The puu gene cluster is found in E. coli and closely related enterobacteria, but this gene cluster is uncommon in other bacterial groups. E. coli and related enterobacteria may have gained the Puu pathway as an adaptation for survival in the mammalian intestine, an environment in which polyamines exist at relatively high concentrations.

PGLN: A newly identified amino phosphoglycolipid species in Thermus thermophilus HB8.

Thermus thermophilus has several minor lipid molecules with structures that have not been described yet. In this study, we identified a new lipid molecule in T. thermophilus HB8 with an amino group at the polar head, by detecting lipid spots with HPTLC and mass spectrometry. The structure of the lipid resembles an amino sugar phospholipid, except for the glucosamine that lacks an acetyl group. We named this amino phosphoglycolipid PGLN, and proposed its synthetic pathway from a precursor, phosphatidyl-glyceric alkylamine. The primary amine structure of PGLN may contribute to high temperature adaptation through electrostatic interactions between the head groups.

Yeast 18 S rRNA is directly involved in the ribosomal response to stringent AUG selection during translation initiation.

In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNA(i)(Met), show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40 S subunits.

Complete Genome Sequence of Thermus thermophilus Strain HB5018, Isolated from Mine Hot Spring in Japan.

We isolated Thermus thermophilus strain HB5018 from Mine Hot Spring in Japan, where the type strain HB8 was isolated nearly half a century ago. The complete genome sequence of HB5018 showed 99.1% average nucleotide identity with HB8, suggesting strict species conservation in the habitat over the past 50 years.

Crystal structure of adenylate kinase from an extremophilic archaeon Aeropyrum pernix with ATP and AMP.

The crystal structure of an adenylate kinase from an extremophilic archaeon Aeropyrum pernix was determined in complex with full ligands, ATP-Mg2+ and AMP, at a resolution of 2.0 Å. The protein forms a trimer as found for other adenylate kinases from archaea. Interestingly, the reacting three atoms, two phosphorus and one oxygen atoms, were located almost in line, supporting the SN2 nucleophilic substitution reaction mechanism. Based on the crystal structure obtained, the reaction coordinate was estimated by the quantum mechanics calculations combined with molecular dynamics. It was found that the reaction undergoes two energy barriers; the steps for breaking the bond between the oxygen and γ-phosphorus atoms of ATP to produce a phosphoryl fragment and creating the bond between the phosphoryl fragment and the oxygen atom of the ß-phosphate group of ADP. The reaction coordinate analysis also suggested the role of amino-acid residues for the catalysis of adenylate kinase.

Crystal structure of (S)-3-O-geranylgeranylglyceryl phosphate synthase from Thermoplasma acidophilum in complex with the substrate sn-glycerol 1-phosphate.

(S)-3-O-Geranylgeranylglyceryl phosphate synthase (GGGPS) catalyzes the initial ether-bond formation between sn-glycerol 1-phosphate (G1P) and geranylgeranyl pyrophosphate to synthesize (S)-3-O-geranylgeranylglyceryl phosphate in the production of an archaeal cell-membrane lipid molecule. Archaeal GGGPS proteins are divided into two groups (group I and group II). In this study, the crystal structure of the archaeal group II GGGPS from Thermoplasma acidophilum (TaGGGPS) was determined at 2.35 Šresolution. The structure of TaGGGPS showed that it has a TIM-barrel fold, the third helix of which is disordered (α3*), and that it forms a homodimer, although a pre-existing structure of an archaeal group II GGGPS (from Methanothermobacter thermautotrophicus) showed a hexameric form. The structure of TaGGGPS showed the precise G1P-recognition mechanism of an archaeal group II GGGPS. The structure of TaGGGPS and molecular-dynamics simulation analysis showed fluctuation of the ß2-α2, α3* and α5a regions, which is predicted to be important for substrate uptake and/or product release by TaGGGPS.

Effects of pH and temperature on the composition of polar lipids in Thermoplasma acidophilum HO-62.

Thermoplasma acidophilum HO-62 was grown at different pHs and temperatures, and its polar lipid compositions were determined. Although the number of cyclopentane rings in the caldarchaeol moiety increased when T. acidophilum was cultured at high temperature, the number decreased at low pHs. Glycolipids, phosphoglycolipids, and phospholipids were analyzed by high-performance liquid chromatography with an evaporative light-scattering detector. The amount of caldarchaeol with more than two sugar units on one side increased under low-pH and high-temperature conditions. The amounts of glycolipids increased and those of phosphoglycolipids decreased under these conditions. The proton permeability of the liposomes obtained from the phosphoglycolipids that contained two or more sugar units was lower than that of the liposomes obtained from the phosphoglycolipids that contained one sugar unit. From these results, we propose the hypothesis that T. acidophilum adapts to low pHs and high temperatures by extending sugar chains on their cell surfaces, as well as by varying the number of cyclopentane rings.

Purification and characterization of geranylgeranylglyceryl phosphate synthase from a thermoacidophilic archaeon, Thermoplasma acidophilum.

We purified a geranylgeranylglyceryl phosphate (GGGP) synthase from Thermoplasma acidophilum by several steps of chromatography. Based on the proteinase-fragment-mass-pattern analysis of the SDS-PAGE band of the partially purified protein, the DNA sequence encoding the protein was identified from the whole genome sequence database of the species. The gene encoding GGGP synthase in T. acidophilum was cloned after PCR amplification of the gene from the genomic DNA. The recombinant enzyme was expressed in Escherichia coli and purified. A single band with a molecular mass of 27 kDa was obtained by SDS-PAGE analysis. The apparent native molecular mass of the enzyme was about 50 kDa based on gel filtration chromatography, suggesting that the enzyme is active as a homodimer. As the GGGP synthase from Methanobacterium thermoautotrophicum has been reported as a pentamer, the enzymes of the two organisms have different oligomeric structures. Other characteristics, including substrate specificity, are similar for the GGGPs of these organisms.

Interaction between 25S rRNA A loop and eukaryotic translation initiation factor 5B promotes subunit joining and ensures stringent AUG selection.

In yeast, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During the last step of translation initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S initiation complex (IC). Malfunctional 60S subunits produced by misfolding or mutation may disrupt the 40S IC stalling on the start codon, thereby altering the stringency of initiation. Using several point mutations isolated by random mutagenesis, here we studied the role of 25S rRNA in start codon selection. Three mutations changing bases near the ribosome surface had strong effects, allowing the initiating ribosomes to skip both AUG and non-AUG codons: C2879U and U2408C, altering the A loop and P loop, respectively, of the peptidyl transferase center, and G1735A, mapping near a Eukarya-specific bridge to the 40S subunit. Overexpression of eIF5B specifically suppressed the phenotype caused by C2879U, suggesting functional interaction between eIF5B and the A loop. In vitro reconstitution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC. Thus, eIF5B interaction with the peptidyl transferase center A loop increases the accuracy of initiation by stabilizing the overall conformation of the 80S initiation complex. This study provides an insight into the effect of ribosomal mutations on translation profiles in eukaryotes.

Random mutagenesis of yeast 25S rRNA identify bases critical for 60S subunit structural integrity and function.

In yeast Saccharomyces cerevisiae, 25S rRNA makes up the major mass and shape of the 60S ribosomal subunit. During translation initiation, the 60S subunit joins the 40S initiation complex, producing the 80S initiation complex. During elongation, the 60S subunit binds the CCA-ends of aminoacyl- and peptidyl-tRNAs at the A-loop and P-loop, respectively, transferring the peptide onto the α-amino group of the aminoacyl-tRNA. To study the role of 25S rRNA in translation in vivo, we randomly mutated 25S rRNA and isolated and characterized seven point mutations that affected yeast cell growth and polysome profiles. Four of these mutations, G651A, A1435U, A1446G and A1587G, change a base involved in base triples crucial for structural integrity. Three other mutations change bases near the ribosomal surface: C2879U and U2408C alter the A-loop and P-loop, respectively, and G1735A maps near a Eukarya-specific bridge to the 40S subunit. By polysome profiling in mmslΔ mutants defective in nonfunctional 25S rRNA decay, we show that some of these mutations are defective in both the initiation and elongation phases of translation. Of the mutants characterized, C2879U displays the strongest defect in translation initiation. The ribosome transit-time assay directly shows that this mutation is also defective in peptide elongation/termination. Thus, our genetic analysis not only identifies bases critical for structural integrity of the 60S subunit, but also suggests a role for bases near the peptidyl transferase center in translation initiation.

The roles of stress-activated Sty1 and Gcn2 kinases and of the protooncoprotein homologue Int6/eIF3e in responses to endogenous oxidative stress during histidine starvation.

In fission yeast, Sty1 and Gcn2 are important protein kinases that regulate gene expression in response to amino acid starvation. The translation factor subunit Int6/eIF3e promotes Sty1-dependent response by increasing the abundance of Atf1, a transcription factor targeted by Sty1. While Gcn2 promotes expression of amino acid biosynthesis enzymes, the mechanism and function of Sty1 activation and Int6/eIF3e involvement during this nutrient stress are not understood. Here we show that mutants lacking sty1(+) or gcn2(+) display reduced viabilities during histidine depletion stress in a manner suppressible by the antioxidant N-acetyl cysteine, suggesting that these protein kinases function to alleviate endogenous oxidative damage generated during nutrient starvation. Int6/eIF3e also promotes cell viability by a mechanism involving the stimulation of Sty1 response to oxidative damage. In further support of these observations, microarray data suggest that, during histidine starvation, int6Δ increases the duration of Sty1-activated gene expression linked to oxidative stress due to the initial attenuation of Sty1-dependent transcription. Moreover, loss of gcn2 induces the expression of a new set of genes not activated in wild-type cells starved for histidine. These genes encode heatshock proteins, redox enzymes, and proteins involved in mitochondrial maintenance, in agreement with the idea that oxidative stress is imposed on gcn2Δ cells. Furthermore, early Sty1 activation promotes rapid Gcn2 activation on histidine starvation. These results suggest that Gcn2, Sty1, and Int6/eIF3e are functionally integrated and cooperate to respond to oxidative stress generated during histidine starvation.

Int6/eIF3e promotes general translation and Atf1 abundance to modulate Sty1 MAPK-dependent stress response in fission yeast.

int-6 is one of the frequent integration sites for mouse mammary tumor viruses. Although its product is the e-subunit of translation initiation factor eIF3, other evidence indicates that it interacts with proteasomes or other proteins to regulate protein stability. Here we report that the fission yeast int6(+) is required for overcoming stress imposed by histidine starvation, using the drug 3-aminotriazole (3AT). Microarray and complementary Northern studies using wild-type, int6Delta or gcn2Delta mutants indicate that 3AT-treated wild-type yeast induces core environmental stress response (CESR) genes in addition to typical general amino acid control (GAAC) genes whose transcription depends on the eIF2 kinase, Gcn2. In agreement with this, Sty1 MAPK and its target transcription factor Atf1, which signal the CESR, are required for overcoming 3AT-induced starvation. We find that Int6 is required for maintaining the basal level of Atf1 and for rapid transcriptional activation of the CESR on 3AT-insult. Pulse labeling experiments indicate that int6Delta significantly slows down de novo protein synthesis. Moreover, Atf1 protein half-life was reduced in int6Delta cells. These effects would account for the compromised Atf1 activity on 3AT-induced stress. Thus, the robust protein synthesis promoted by intact eIF3 appears to be a part of the requisites for sound Sty1 MAPK-dependent signaling governed by the activity of the Atf1 transcription factor.

An actin homolog of the archaeon Thermoplasma acidophilum that retains the ancient characteristics of eukaryotic actin.

Actin, a central component of the eukaryotic cytoskeleton, plays a crucial role in determining cell shape in addition to several other functions. Recently, the structure of the archaeal actin homolog Ta0583, isolated from the archaeon Thermoplasma acidophilum, which lacks a cell wall, was reported by Roeben et al. (J. Mol. Biol. 358:145-156, 2006). Here we show that Ta0583 assembles into bundles of filaments similar to those formed by eukaryotic actin. Specifically, Ta0583 forms a helix with a filament width of 5.5 nm and an axial repeating unit of 5.5 nm, both of which are comparable to those of eukaryotic actin. Eukaryotic actin shows a greater resemblance to Ta0583 than to bacterial MreB and ParM in terms of polymerization characteristics, such as the requirement for Mg(2+), critical concentration, and repeating unit size. Furthermore, phylogenetic analysis also showed a closer relationship between Ta0583 and eukaryotic actin than between MreB or ParM and actin. However, the low specificity of Ta0583 for nucleotide triphosphates indicates that Ta0583 is more primitive than eukaryotic actin. Taken together, our results suggest that Ta0583 retains the ancient characteristics of eukaryotic actin.

Processing of the tail lysozyme (gp5) of bacteriophage T4.

The processing site of gp5 has been determined to be between residues Val-390 and His-391, instead of Ser-351 and Ala-352 as previously reported (H. Kanamaru, N. C. Gassner, N. Ye, S. Takeda, and F. Arisaka, J. Bacteriol. 181:2739-2744). Moreover, the maturation of gp5 is abolished by null mutations in other hub genes, indicating that cleavage requires the interactions of several baseplate proteins.

Effects of a squalene epoxidase inhibitor, terbinafine, on ether lipid biosyntheses in a thermoacidophilic archaeon, Thermoplasma acidophilum.

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-(14)C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.

Characterization of the precursor of tetraether lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum.

Polar lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum was analyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [(14)C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chromatography. Lipids labeled with [(14)C]mevalonic acid, [(14)C]glycerol, and [(32)P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophosphatidylcaldarchaetidylglycerol, caldarchaetidylglycerol, and beta- l-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetraether lipid molecule is synthesized by head-to-head condensation of two molecules of archaetidylglycerol and that a sugar group of tetraether phosphoglycolipid is expected to attach to the tetraether lipid core after head-to-head condensation in T. acidophilum. A precursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The relative height of the peak m/z 806 decreased after removal of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form.