Mitochondria-encoded ND (NADH dehydrogenase) subunits, as components of the hydrophobic part of complex I, are essential for NADH:ubiquinone oxidoreductase activity. Mutations or lack of expression of these subunits have significant pathogenic consequences in humans. However, the way these events affect complex I assembly is poorly documented. To understand the effects of particular mutations in ND subunits on complex I assembly, we studied four human cell lines: ND4 non-expressing cells, ND5 non-expressing cells, and rho degrees cells that do not express any ND subunits, in comparison with normal complex I control cells. In control cells, all the seven analysed nuclear-encoded complex I subunits were found to be attached to the mitochondrial inner membrane, except for the 24 kDa subunit, which was nearly equally partitioned between the membranes and the matrix. Absence of a single ND subunit, or even all the seven ND subunits, caused no major changes in the nuclear-encoded complex I subunit content of mitochondria. However, in cells lacking ND4 or ND5, very low amounts of 24 kDa subunit were found associated with the membranes, whereas most of the other nuclear-encoded subunits remained attached. In contrast, membrane association of most of the nuclear subunits was significantly reduced in the absence of all seven ND proteins. Immunopurification detected several subcomplexes. One of these, containing the 23, 30 and 49 kDa subunits, also contained prohibitin. This is the first description of prohibitin interaction with complex I subunits and suggests that this protein might play a role in the assembly or degradation of mitochondrial complex I.

Several mutations in the mitochondrially encoded cytochrome b have been reported in patients. To characterize their effect, we introduced six "human" mutations, namely G33S, S152P, G252D, Y279C, G291D, and ⌬252-259 in the highly similar yeast cytochrome b. G252D showed wild type behavior in standard conditions. However, Asp-252 may interfere with structural lipid and, in consequence, destabilize the enzyme assembly, which could explain the pathogenicity of the mutation. The mutations G33S, S152P, G291D, and ⌬252-259 were clearly pathogenic. They caused a severe decrease of the respiratory function and altered the assembly of the iron-sulfur protein in the bc 1 complex, as observed by immunodetection. Suppressor mutations that partially restored the respiratory function impaired by S152P or G291D were found in or close to the hinge region of the iron-sulfur protein, suggesting that this region may play a role in the stable binding of the subunit to the bc 1 complex. Y279C caused a significant decrease of the bc 1 function and perturbed the quinol binding. The EPR spectra showed an altered signal, indicative of a lower occupancy of the Q o site. The effect of human mutation of residue 279 was confirmed by another change, Y279A, which had a more severe effect on Q o site properties. Thus by using yeast as a model system, we identified the molecular basis of the respiratory defect caused by the disease mutations in cytochrome b.The mitochondrial bc 1 complex is a membrane-bound enzyme that catalyzes the transfer of electrons from ubiquinol to cytochrome c and couples this electron transfer to vectorial proton translocation across the inner mitochondrial membrane. The enzyme exists as a functional dimer, consisting of 10 or 11 polypeptides in the eukaryotic monomeric subunit. One subunit, cytochrome b, is encoded by the mitochondrial genome, whereas the others are nuclearly encoded. Redox prosthetic groups are located within three subunits: cytochrome c 1 and the iron-sulfur protein (ISP), 1 which are membrane proteins with large, hydrophilic domains, and cytochrome b, a predominantly hydrophobic protein consisting of eight transmembrane helices that contains two b-type hemes (b l and b h ) and forms the two quinol binding sites: Q o (site of quinol oxidation) and Q i (site of quinol reduction), located on opposite sides of the membrane. The catalytic mechanism of the bc 1 complex is essentially described by Mitchell's Q-cycle model. A quinol molecule binds at the Q o -site, is deprotonated, and transfers one electron through the "high potential" electron transfer chain consisting of the [2Fe-2S] cluster of the ISP and the c-type heme of cytochrome c 1 to the soluble acceptor, cytochrome c. Following a bifurcated pathway, a second electron is transferred across the membrane by the "low potential" pathway formed from hemes b l and b h and delivered to quinone bound at the Q i site, forming a stable semiquinone.

Background Biosimilars have been used for 15 years in the European Union (EU), and have been shown to reduce costs and increase access to important biological medicines. In spite of their considerable exposure and excellent safety record, many prescribers still have doubts on the safety and interchangeability of biosimilars, especially monoclonal antibodies (mAbs) and fusion proteins. Objectives The aim of this study was to analyse the short-and long-term safety and interchangeability data of biosimilar mAbs and fusion proteins to provide unbiased information to prescribers and policy makers. Methods Data on the safety, immunogenicity and interchangeability of EU-licensed mAbs and fusion proteins were examined using European Public Assessment Reports (EPARs) and postmarketing safety surveillance reports from the European Medicines Agency (EMA). As recent biosimilar approvals allow self-administration by patients by the subcutaneous route, the administration devices were also analyzed. Results Prelicensing data of EPARs (six different biosimilar adalimumabs, three infliximabs, three etanercepts, three rituximabs, two bevacizumabs, and six trastuzumabs) revealed that the frequency of fatal treatment-emergent adverse events (TEAEs), TEAEs leading to discontinuation of treatment, serious adverse events (SAEs), and main immune-mediated adverse events (AEs) were comparable between the biosimilars and their reference products. The availability of new biosimilar presentations and administration devices may add to patient choice and be an emerging factor in the decision to switch patients. Analysis of postmarketing surveillance data covering up to 7 years of follow-up did not reveal any biosimilar-specific adverse effects. No product was withdrawn for safety reasons. This is in spite of considerable exposure to biosimilars in treatmentnaïve patients and in patients switched from the reference medicinal product to the biosimilar. Analysis of data from switching studies provided in regulatory submissions showed that single or multiple switches between the originator and its biosimilar versions had no negative impact on efficacy, safety or immunogenicity. Conclusions In line with previous reports of prelicensing studies of biosimilar mAbs and etanercepts, this study demonstrated comparable efficacy, safety, and immunogenicity compared with the reference products. This is the first study to comprehensively analyze postmarketing surveillance data of the biosimilar mAbs and etanercept. An analysis of more than 1 million patient-treatment years of safety data raised no safety concerns. Based on these data, we argue that biosimilars approved in the EU are highly similar to and interchangeable with their reference products. Thus, additional systematic switch studies are not required to support the switching of patients.