A THEMIS:SHP1 complex promotes T‐cell survival

THEMIS is critical for conventional T‐cell development, but its precise molecular function remains elusive. Here, we show that THEMIS constitutively associates with the phosphatases SHP1 and SHP2. This complex requires the adapter GRB2, which bridges SHP to THEMIS in a Tyr‐phosphorylation‐independent fashion. Rather, SHP1 and THEMIS engage with the N‐SH3 and C‐SH3 domains of GRB2, respectively, a configuration that allows GRB2‐SH2 to recruit the complex onto LAT. Consistent with THEMIS‐mediated recruitment of SHP to the TCR signalosome, THEMIS knock‐down increased TCR‐induced CD3‐ζ phosphorylation, Erk activation and CD69 expression, but not LCK phosphorylation. This generalized TCR signalling increase led to augmented apoptosis, a phenotype mirrored by SHP1 knock‐down. Remarkably, a KI mutation of LCK Ser59, previously suggested to be key in ERK‐mediated resistance towards SHP1 negative feedback, did not affect TCR signalling nor ligand discrimination in vivo. Thus, the THEMIS:SHP complex dampens early TCR signalling by a previously unknown molecular mechanism that favours T‐cell survival. We discuss possible implications of this mechanism in modulating TCR output signals towards conventional T‐cell development and differentiation.     

Rapid kinetics of tyrosyl radical formation and heme redox state changes in prostaglandin H synthase-1 and -2.

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34-35 G) was formed by 4 ms, followed by transition to a wide singlet (33-34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25-26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.     

Prostaglandin H synthase and hydroperoxides: peroxidase reaction and inactivation kinetics

The reaction kinetics of the peroxidase activity of prostaglandin H synthase have been examined with 15-hydroperoxyeicosatetraenoic acid and hydrogen peroxide as substrates and tetramethylphenylenediamine as cosubstrate. The apparent Km and Vmax values for both hydroperoxides were found to increase linearly with the cosubstrate concentration. The overall reaction kinetics could be interpreted in terms of an initial reaction of the synthase with hydroperoxide to form an intermediate equivalent to horseradish peroxidase Compound I, followed by reduction of this intermediate by cosubstrate to regenerate the resting enzyme. The rate constants estimated for the generation of synthase Compound I were 7.1 X 10(7) M-1 s-1 with the lipid hydroperoxide and 9.1 X 10(4) M-1 s-1 with hydrogen peroxide. The rate constants estimated for the rate-determining step in the regeneration of resting enzyme by cosubstrate were 9.2 X 10(6) M-1 s-1 in the case of the reaction with lipid hydroperoxide and 3.5 X 10(6) M-1 s-1 in the case of reaction with hydrogen peroxide. The intrinsic affinities of the synthase peroxidase for substrate (Ks) were estimated to be on the order of 10(-8) M for lipid hydroperoxide and 10(-5) M for hydrogen peroxide. These affinities are quite similar to the reported affinities of the synthase for these hydroperoxides as activators of the cyclooxygenase. The peroxidase activity was found to be progressively inactivated during the peroxidase reaction. The rate of inactivation of the peroxidase was increased by increases in hydroperoxide level, and decreased by increases in peroxidase cosubstrate. The inactivation of the peroxidase appeared to occur by a hydroperoxide-dependent process, originating from synthase Compound I or Compound II.     

Cyclooxygenase reaction mechanism of prostaglandin H synthase from deuterium kinetic isotope effects

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS) is thought to involve a multistep mechanism with several radical intermediates. The proposed mechanism begins with the transfer of the C13 pro-(S) hydrogen atom from the substrate arachidonic acid (AA) to the Tyr385 radical in PGHS, followed by oxygen insertion and several bond rearrangements. The importance of the hydrogen-transfer step to controlling the overall kinetics of cyclooxygenase catalysis has not been directly examined. We quantified the non-competitive primary kinetic isotope effect (KIE) for both PGHS-1 and -2 using several deuterated AAs, including 13-pro-(S) d-AA, 13,13-d₂-AA and 10, 10, 13,13-d₄-AA. The primary KIE for steady-state cyclooxygenase catalysis, ^Dk_cat, ranged between 1.8 and 2.3 in oxygen electrode measurements. The intrinsic KIE of AA radical formation by C13 pro-(S) hydrogen abstraction in PGHS-1 was estimated to be 1.9-2.3 using rapid freeze-quench EPR kinetic analysis of anaerobic reactions and computer modeling to a mechanism that includes a slow formation of a pentadienyl AA radical and a rapid equilibration of the AA radical with a tyrosyl radical, NS1c. The observation of similar values for steady-state and pre-steady state KIEs suggests that hydrogen abstraction is a rate-limiting step in cyclooxygenase catalysis. The large difference of the observed KIE from that of plant lipoxygenases indicates that PGHS and lipoxygenases have very different mechanisms of hydrogen transfer.     

Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure

Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40–80 °C) maximum in situ temperatures (∼23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40–60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii^T (∼96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii^Tand D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.