Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

Properdin enhances complement-mediated opsonization of targeted cells and particles for immune clearance. Properdin occurs as dimers, trimers and tetramers in human plasma, which recognize C3b-deposited surfaces, promote formation, and prolong the lifetime of C3bBb-enzyme complexes that convert C3 into C3b, thereby enhancing the complement-amplification loop. Here, we report crystal structures of monomerized properdin, which was produced by co-expression of separate N- and C-terminal constructs that yielded monomer-sized properdin complexes that stabilized C3bBb. Consistent with previous low-resolution X-ray and EM data, the crystal structures revealed ring-shaped arrangements that are formed by interactions between thrombospondin type-I repeat (TSR) domains 4 and 6 of one protomer interacting with the N-terminal domain (which adopts a short transforming-growth factor B binding protein-like fold) and domain TSR1 of a second protomer, respectively. Next, a structure of monomerized properdin in complex with the C-terminal domain of C3b showed that properdin-domain TSR5 binds along the C-terminal a-helix of C3b, while two loops, one from domain TSR5 and one from TSR6, extend and fold around the C3b C-terminus like stirrups. This suggests a mechanistic model in which these TSR5 and TSR6 "stirrups" bridge interactions between C3b and factor B or its fragment Bb, and thereby enhance formation of C3bB pro-convertases and stabilize C3bBb convertases. In addition, properdin TSR6 would sterically block binding of the protease factor I to C3b, thus limiting C3b proteolytic degradation. The presence of a valine instead of a third tryptophan in the canonical Trp-ladder of TSR domains in TSR4 allows a remarkable ca. 60 degrees-domain bending motion of TSR4. Together with variable positioning of TSR2 and, putatively, TSR3, this explains the conformational flexibility required for properdin to form dimers, trimers, and tetramers. In conclusion, the results indicate that binding avidity of oligomeric properdin is needed to distinguish surface-deposited C3b molecules from soluble C3b or C3 and suggest that properdin-mediated interactions bridging C3b-B and C3b-Bb enhance affinity, thus promoting convertase formation and stabilization. These mechanisms explain the enhancement of complement-mediated opsonization of targeted cells and particle for immune clearance

Gender differences in hyperthermia and regional 5-HT and 5-HIAA depletion in the brain following MDMA administration in rats

In the present research the role of gender in MDMA-induced hyperthermia and serotonin depletion is studied by injecting male and female male rats with MDMA or saline 3 times (i.p.) with 3 h interval at dosages of 0.3, 1, 3 or 9 mg/kg at an ambient temperature of 25 °C. The acute hyperthermia following the higher dosages was much stronger in males than in females. After the highest dose, body temperature was even raised for several days. This effect was particularly present in males where nocturnal hyperthermia persisted the whole 4-week period of sampling. Despite the differences in the acute hyperthermic response, no significant gender differences were found in 5-HT depletion 4 weeks after MDMA (9 mg/kg) administration. A striking difference was present, however, in the concentration of the 5-HT metabolite 5-HIAA after MDMA administration. In males 5-HIAA levels decreased, whereas in females this metabolite was hardly affected, suggesting a lasting increase in 5-HT turnover in females following drug administration. When genders were matched for their acute physiological hyperthermic response by repeated injection of 9 mg/kg in female rats and 6 mg/kg in male rats, 5-HT depletion was only present in females. In this experiment with matched acute physiological responses 5-HIAA levels also decreased much stronger in males, suggesting an increased 5-HT turnover in females 4 weeks after MDMA administration. In conclusion, although male rats are clearly more susceptible for the acute as well as the lasting hyperthermic effects of MDMA than females, this is not reflected in levels of 5-HT depletion following administration of similar dosages of the drug. This may indicate that, in case of a similar thermogenic response, females have a higher 5-HT neurotoxicity following MDMA than males.

Long-term neurobiological consequences of ecstasy: A role for pre-existing trait-like differences in brain monoaminergic functioning?

This study investigated whether trait-like differences in brain monoaminergic functioning relate to differential vulnerability for the long-term neurochemical depletion effects of MDMA. Genetically selected aggressive (SAL) and non-aggressive (LAL) house-mice differing in baseline serotonergic and dopaminergic neurotransmission were administered MDMA. An acute binge-like MDMA injection protocol (three times, using either of the dosages of 0, 5, 10 and 20 mg/kg i.p. with 3 h interval) was employed. Three and 28 days after treatment with MDMA induced a dose-dependent depletion of striatal dopamine and its metabolites that did not differ between SAL and LAL mice. Similarly, the dose-dependent MDMA-induced serotonergic depletion did not differ between lines 3 days after treatment. Interestingly, 28 days after MDMA in LAL mice, 5-HT and 5-HIAA levels were still significantly depleted after treatment with 3×10 mg/kg, while in SAL mice 5-HT depletion was only seen after the highest dosage. Surprisingly, LAL mice did not show any long-term 5-HT depletion after treatment with the highest dose. In conclusion, only LAL mice are able to restore initial severe loss of MDMA-evoked 5-HT and 5-HIAA levels. SAL and LAL mice are differentially susceptible for the long-term but not short-term MDMA-induced serotonergic depletion in the striatum. The differentiation between both lines in the long-term striatal serotonergic response to MDMA seems to depend on the capacity of the brain to adapt to the short-term depletion of monoaminergic levels and may somehow be related to individual, trait-like characteristics of brain monoaminergic systems.

Social stress during adolescence in Wistar rats induces social anxiety in adulthood without affecting brain monoaminergic content and activity

Adolescence has been described as an important period to acquire social competences required for adult life. It has been suggested that early stress experiences could affect the development of the brain at different levels. These changes in the brain during adolescence may be related with the development of psychopathologies such as depression and social anxiety in adulthood. In the first experiment, we examined long-term effects of repeated social stress during adolescence on adult social approach–avoidance behavior. For that purpose, adolescent male Wistar rats were exposed twice at postnatal day (Pnd) 45 and Pnd48 to the resident–intruder paradigm followed by three times psychosocial threat with the same resident. Three weeks after the last psychosocial threat experience the animals were behaviorally tested in a social approach–avoidance test. Socially stressed animals spent less time in the interaction zone with an unfamiliar male adult rat. These data suggest that animals exposed to social stress during adolescence show a higher level of social anxiety in adulthood. In the second experiment, we investigated whether these long-term effects of social stress during adolescence on behavior draw a parallel with changes in brain monoamine content, biosynthesis and turnover. Using the same experimental design as in the first experiment, HPLC analysis of various brain regions showed that there were no differences in monoamine content, monoamine biosynthesis and monoamines activity in the prefrontal cortex, hippocampus, hypothalamus and striatum in adulthood. These results indicate that long-lasting changes in social behavior following social stress during adolescence are not accompanied by changes in brain monoamine content, biosynthesis and turnover.

Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

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Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

Properdin enhances complement-mediated opsonization of targeted cells and particles for immune clearance. Properdin occurs as dimers, trimers and tetramers in human plasma, which recognize C3b-deposited surfaces, promote formation, and prolong the lifetime of C3bBb-enzyme complexes that convert C3 into C3b, thereby enhancing the complement-amplification loop. Here, we report crystal structures of monomerized properdin, which was produced by co-expression of separate N- and C-terminal constructs that yielded monomer-sized properdin complexes that stabilized C3bBb. Consistent with previous low-resolution X-ray and EM data, the crystal structures revealed ring-shaped arrangements that are formed by interactions between thrombospondin type-I repeat (TSR) domains 4 and 6 of one protomer interacting with the N-terminal domain (which adopts a short transforming-growth factor B binding protein-like fold) and domain TSR1 of a second protomer, respectively. Next, a structure of monomerized properdin in complex with the C-terminal domain of C3b showed that properdin-domain TSR5 binds along the C-terminal a-helix of C3b, while two loops, one from domain TSR5 and one from TSR6, extend and fold around the C3b C-terminus like stirrups. This suggests a mechanistic model in which these TSR5 and TSR6 "stirrups" bridge interactions between C3b and factor B or its fragment Bb, and thereby enhance formation of C3bB pro-convertases and stabilize C3bBb convertases. In addition, properdin TSR6 would sterically block binding of the protease factor I to C3b, thus limiting C3b proteolytic degradation. The presence of a valine instead of a third tryptophan in the canonical Trp-ladder of TSR domains in TSR4 allows a remarkable ca. 60 degrees-domain bending motion of TSR4. Together with variable positioning of TSR2 and, putatively, TSR3, this explains the conformational flexibility required for properdin to form dimers, trimers, and tetramers. In conclusion, the results indicate that binding avidity of oligomeric properdin is needed to distinguish surface-deposited C3b molecules from soluble C3b or C3 and suggest that properdin-mediated interactions bridging C3b-B and C3b-Bb enhance affinity, thus promoting convertase formation and stabilization. These mechanisms explain the enhancement of complement-mediated opsonization of targeted cells and particle for immune clearance