A reductase/isomerase subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) carries an NADPH and is involved in the biogenesis of the complex.

Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I. The bacterial complex I consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. One of these extra subunits with a molecular mass of 40 kDa belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. We deleted this subunit in Neurospora crassa by gene disruption. In the mutant nuo 40, a complex I lacking the 40 kDa subunit is assembled. The mutant complex I does not contain tightly bound NADPH present in wild-type complex I. This NADPH cofactor is not connected to the respiratory electron pathway of complex I. The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-type complex I, one flavin mononucleotide and four iron-sulfur clusters detectable by electron paramagnetic resonance spectroscopy. In the mutant complex these groups are all readily reduced by NADH. However, the mutant complex is not capable of reducing ubiquinone. A recently described redox group identified in wild-type complex I by UV-visible spectroscopy is not detectable in the mutant complex. We propose that the reductase/isomerase subunit with its NADPH cofactor takes part in the biosynthesis of this new redox group.

CXCR4 Antagonists: A Screening Strategy for Identification of Functionally Selective Ligands.

The CXC chemokine receptor 4 (CXCR4) is a widely expressed G protein-coupled receptor implicated in several diseases. In cancer, an increased number of surface CXCR4 receptors, in parallel with aberrant signaling, have been reported to influence several aspects of malignancy progression. CXCR4 activation by the specific ligand C-X-C motif chemokine 12 (CXCL12) induces several intracellular signaling pathways that have been selectively related to malignancy depending on the tissue or cell type. We developed a panel of CXCR4 screening assays investigating Gα(i)-mediated cyclic adenosine monophosphate modulation, ß-arrestin recruitment, and receptor internalization. All of the assays were set up in recombinant cells and were used to test four reported CXCR4 antagonists. Consequently, a set of hit compounds, deriving from a screening campaign of a 30,000-small-molecule internal library, was profiled with the different assays. We identified several compounds showing a pathway-selective activity: antagonists on a Gα(i)-dependent pathway; antagonists on both the ß-arrestin and Gα(i)-dependent pathways, some of which induce receptor internalization; and compounds with an antagonist behavior in all of the readouts. The identified biased antagonists induce different functional states on CXCR4 and preferentially affect specific downstream responses from the activated receptor, thus providing an improved therapeutic profile for correction of CXCR4 abnormal signaling.

Disruption of the gene encoding the NADH-binding subunit of NADH: ubiquinone oxidoreductase in Neurospora crassa. Formation of a partially assembled enzyme without FMN and the iron-sulphur cluster N-3.

In this study, the gene of the 51-kDa NADH-binding subunit of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) in Neurospora crassa was inactivated by homologous replacement with a defective gene copy. The resulting mutant, nuo51, lacks the 51-kDa subunit and shows no complex I activity but still grows at one third of the wild-type growth rate. The enzyme activity of the alternative NADH:ubiquinone oxidoreductase(s) is increased twofold while the activities of the other mitochondrial respiratory enzymes are normal. Complex I is almost completely assembled except for the NADH-binding subunit and still possesses three out of the four EPR-detectable iron-sulphur clusters. Since the deleted subunit contains the sequence motif for one tetranuclear iron-sulphur cluster, the missing cluster N-3 is considered to be bound to this subunit.

Attempts to define distinct parts of NADH:ubiquinone oxidoreductase (complex I).

The NADH:ubiquinone oxidoreductase (complex I) is made up of a peripheral part and a membrane part. The two parts are arranged perpendicular to each other and give the complex an unusual L-shaped structure. The peripheral part protrudes into the matrix space and constitutes the proximal segment of the electron pathway with the NADH-binding site, the FMN and at least three iron-sulfur clusters. The membrane part constitutes the distal segment of the electron pathway with at least one iron-sulfur cluster and the ubiquinone-binding site. Both parts are assembled separately and relationships of the major structural modules of the two parts with different bacterial enzymes suggest, that both parts also emerged independently in evolution. This assumption is further supported by the conserved order of bacterial complex I genes, which correlates with the topological arrangement of the corresponding subunits in the two parts of complex I.

Expression of factor I-resistant mutants of the human complement component C3 in heterologous systems.

Complement plays a major role in hyperacute rejection of xenografts. In order to overcome this, we are developing, by minimal mutagenesis, a modified C3 molecule that, like cobra venom factor (CVF), escapes normal complement regulatory processes and inhibits complement-mediated responses by systemic depletion of C3. Unlike CVF, this protein should have little or no immunogenicity and be suitable for repeat administrations. As an initial step in this process, we have modified human C3 to make it resistant to inactivation by factor I. The factor I resistant C3 is capable of forming an active C3 convertase. Preincubation with normal human serum abrogated subsequent complement-mediated cytolysis by both the classical and alternative pathways, while wild-type (wt) C3 was inactive. The modified human C3 also blocked complement activity of guinea-pig serum. For economical and rapid production, we have developed expression of recombinant C3 wt and mutant proteins in the Baculovirus system. Large quantities are also being produced from stably transfected CHO cell lines. In addition, we have developed a fast C3 purification method by engineering a 6XHIS tag into the C3a portion of the molecule, thereby avoiding the need for subsequent separation of the tag from active C3b molecules.