Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the F _O F ₁ ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca ²⁺ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca ²⁺ . Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca ²⁺ , addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg ²⁺ /ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

In GPxs, the redox-active Se or S, is at hydrogen bonding distance from Gln and Trp residues that contribute to catalysis. From sequence homology of >400 sequences and modeling of the DmGPx as a paradigm, Asn136 emerged as a fourth essential component of the active site. Mutational substitution of Asn136 by His, Ala, or Asp results in a dramatic decline of specific activity. Kinetic analysis indicates that k(+1), the rate constant for the oxidation of the enzyme, decreases by two to three orders of magnitude, whereas the reductive steps characterized by k'(+2) are less affected. Accordingly, MS/MS analysis shows that in Asn136 mutants, the peroxidatic Cys45 stays largely reduced also in the presence of a hydroperoxide, whereas in the wild-type enzyme, it is oxidized, forming a disulfide with the resolving Cys. Computational calculation of pK(a) values indicates that the residues facing the catalytic thiol, Asn136, Gln80, and, to a lesser extent Trp135, contribute to the dissociation of the thiol group, Asn136 being most relevant. These data disclose that the catalytic site of GPxs has to be redrawn as a tetrad, including Asn136, and suggest a mechanism accounting for the extraordinary catalytic efficiency of GPxs.

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils ofβ₂-microglobulin (β₂-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, β₂-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology ofβ₂-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in β₂-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role inβ₂-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-l-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-l-lysine is dissolved in solution with β₂-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as β₂-m, that decay on moving away from the fiber surface.