Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study

: Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH3-S-CoM) with coenzyme B (H-S-CoB) to CH4 and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCRred1 both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCRred1 signal is partially converted into the rhombic EPR signal MCRred2. To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic 1H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCRred2 stat

Stability and Cu(II) Binding of Prion Protein Variants Related to Inherited Human Prion Diseases

All inherited forms of human prion diseases are linked with mutations in the prion protein (PrP) gene. Here we have investigated the stability and Cu(II) binding properties of three recombinant variants of murine full-length PrP(23–231)-containing destabilizing point mutations that are associated with human Gerstmann-Sträussler-Scheinker disease (F198S), Creutzfeld-Jakob disease (E200K), and fatal familial insomnia (D178N) by electron paramagnetic resonance and circular dichroism spectroscopy. Furthermore, we analyzed the variants H140S, H177S, and H187S of the isolated C-terminal domain of murine PrP, mPrP(121–231), to test a role of the histidine residues in Cu(II) binding. The F198S and E200K variants of PrP(23–231) differed in Cu(II) binding from the wild-type mPrP(23–231). However, circular dichroism spectroscopy indicated that the variants and the wild type did not undergo conformational changes in the presence of Cu(II). The D178N variant showed a high tendency to aggregate at pH 7.4 both with and without Cu(II). At lower pH values, it showed the same Cu(II) binding behavior as the wild type. The analysis allowed for a better location of the Cu(II) binding sites in the C-terminal part of the protein. Our present data indicate that hereditary forms of prion diseases cannot be rationalized on the basis of altered Cu(II) binding or mutation-induced protein destabilization alone

HIV-1 induces NALP3-inflammasome expression and interleukin-1\u3b2 secretion in dendritic cells from healthy individuals but not from HIV-positive patients.

OBJECTIVE: NALP3-inflammasome is an innate mechanism, alternative to type-1 interferon, which is able to recognize nucleic acids and viruses in the cytoplasm and to induce pro-inflammatory response. Here, we hypothesized the involvement of inflammasome in the early defense against HIV-1 and in the full maturation of dendritic cells: for this, we evaluated the response of dendritic cells pulsed with HIV-1 in terms of inflammasome activation in healthy donors. Moreover, inflammasome response to HIV was evaluated in HIV-infected individuals. DESIGN AND METHODS: Monocyte-derived dendritic cells isolated from 20 healthy individuals (HC-DC) and 20 HIV-1-infected patients (HIV-DC) were pulsed with alditrithiol-2-inactivated HIV-1. We then analyzed inflammasome genes expression and interleukin-1\u3b2 (IL-1\u3b2) secretion. RESULTS: In HC-DC, HIV-1 induced higher NLRP3/NALP3 mRNA expression compared with other inflammasome genes such as NALP1/NLRP1 or IPAF/NLRC4 (P < 0.001). This augmented expression was accompanied by CASP1-increased and IL1B-increased mRNA levels and by a significant increment of IL-1\u3b2 secretion (P < 0.05). Otherwise, HIV-1 failed to activate inflammasome and cytokine production in HIV-DC. HIV-DC showed an increased NLRP3/NALP3 basal expression, suggesting a chronic inflammatory profile of patients' immune cells. CONCLUSION: HIV-1 was able to induce a NALP3-inflammasome response in healthy individuals, indicating that this inflammasome could play a role in the first steps of HIV-1 infection; the consequent inflammatory process may be important for directing host immune response against the virus and/or disease progression. HIV-DC seemed to be chronically activated, but unresponsive against pathogens. Our findings could be of interest considering the ongoing research about dendritic cell manipulation and therapeutic strategies for AIDS involving dendritic cell-based immune-vaccines