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Mitochondrial dysfunction is an important cause for neonatal liver disease. Disruption of genes encoding oxidative phosphorylation (OXPHOS) components usually causes embryonic lethality, and thus few disease models are available. We developed a mouse model for GRAC-ILE syndrome, a neonatal mitochondrial disease with liver and kidney involvement, caused by a homozygous BCS1L mutation (232A>G). This gene encodes a chaperone required for incorporation of Rieske iron-sulfur protein (RISP) into complex III of respiratory chain. Homozygous mutant mice after 3 weeks of age developed striking similarities to the human disease: growth failure, hepatic glycogen depletion, steatosis, fibrosis, and cirrhosis, as well as tubulopathy, complex III deficiency, lactacidosis, and short lifespan. BCS1L was decreased in whole liver cells and isolated mitochondria of mutants at all ages. RISP incorporation into complex III was diminished in symptomatic animals; however, in young animals complex III was correctly assembled. Complex III activity in liver, heart, and kidney of symptomatic mutants was decreased to 20%, 40%, and 40% of controls, respectively, as demonstrated with electron flux kinetics through complex III. In high-resolution respirometry, CIII dysfunction resulted in decreased electron transport capacity through the respiratory chain under maximum substrate input. Complex I function, suggested to be dependent on a functional complex III, was, however, unaffected. Conclusion: We present the first viable model of complex III deficiency mimicking a human mitochondrial disorder. Incorporation of RISP into complex III in young homozygotes suggests another complex III assembly factor during early ontogenesis. The development of symptoms from about 3 weeks of age provides a convenient time window for studying the pathophysiology and treatment of mitochondrial hepatopathy and OXPHOS dysfunction in general. (HEPATOLOGY 2011;53:437-447) R espiratory chain disorders are found in at least 1:5,000 live births. 1 Mitochondrial hepatopathies present at an early age and are found in 10%-20% of patients with respiratory chain defects. 2 Oxidative phosphorylation (OXPHOS) is dependent on the five respiratory chain complexes as well as assembly factors, cofactors, and electron carriers like cytochrome c and ubiquinone. 3 Nuclear DNA genes influencing OXPHOS encode either subunits of the complexes, assembly (ancillary) factors, proteins affecting maintenance and expression of mitochondrial DNA, or proteins related to mitochondrial dynamics. 3,4 One of the ancillary factors is BCS1L, the only known assembly factor for complex III. 5 It was originally identified in yeast as an adenosine triphosphate (ATP)-dependent chaperone, essential for the incorporation of Rieske iron-sulfur protein (RISP) in the last steps of complex III assembly. 6-8 More than 20Abbreviations: BN-PAGE, blue native polyacryl amide electrophoresis; FCCP, the protonophore and uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone; H&E, hematoxylin-eosin; ORO, Oil-R...

SummaryAll virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type-specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen-binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogenbinding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.

Many cells express receptors for plasminogen (Pg), although the responsible molecules in most cases are poorly defined. In contrast, the group A streptococcal surface protein PAM contains a domain with two 13-amino acid residue long repeated sequences (a1 and a2) responsible for Pg binding. Here we identify the region in Pg that interacts with PAM. A radiolabeled proteolytic plasminogen fragment containing the first three kringles (K1-K3) interacted with streptococci expressing PAM or a chimeric surface protein harboring the a1a2 sequence. In contrast, plasminogen fragments containing kringle 4 or kringle 5 and the activable serine proteinase domain failed to bind to PAM-expressing group A streptococci. A synthetic and a recombinant polypeptide containing the a1a2 sequence both bound to immobilized recombinant K2 (rK2) but not to rK1 or rK3. The interaction between the a repeat region and rK2 was reversible, and rK2 completely blocked the binding of Pg to the a1a2 region. The binding of the a repeat containing polypeptide to K2 occurred with an equilibrium association constant of 4.5 ؋ 10 M ؊1, as determined by surface plasmon resonance, a value close to that (1.6 ؋ 10 7 M ؊1 ) calculated for the a1a2-Pg interaction. Inhibition experiments suggested involvement of the lysine-binding site of K2 in the interaction. These data demonstrate that K2 contains the major Pg-binding site for PAM, providing the first well defined example of an interaction between an internal Pg-binding region in a protein and a single kringle domain.The plasma glycoprotein plasminogen (Pg) 1 is a single-chain 92-kDa precursor for the broad spectrum serine proteinase plasmin (1, 2) (see Fig. 1A). In vivo, the tissue-type and urokinase-type plasminogen activators convert the zymogen into the two-chain proteinase by cleavage of a single peptide bond (Arg 561 -Val 562 ). Activation can also be achieved by some bacterial proteins, such as streptokinase from streptococci (1, 2). Plasmin plays a key role in fibrinolysis (1-3) but also participates in several other physiological and pathophysiological processes, including wound healing, tissue penetration of cancer cells, neuronal cell death, and bacterial dissemination (4 -8).The activable serine proteinase domain is located in the COOH-terminal third of Pg. The NH 2 -terminal two-thirds of Pg contains an 8-kDa preactivation peptide and five characteristic kringle domains (K1-K5), each ϳ9 kDa. The kringles mediate interactions with multiple ligands, including fibrin, the primary target of Pg, and ␣ 2 -plasmin inhibitor, its principal regulator (1, 2). The recognition events depend upon interactions between lysine-binding sites in the kringles and exposed COOH-terminal lysines in the ligands. Lysine analogues, such as 6-aminohexanoic acid (6-AHA), mimic COOH-terminal lysines in the interaction with kringles and the structural basis of the interactions between some kringles, particularly K1 and K4, and 6-AHA has been disclosed (9 -12). The affinity of the different kringles for lysine or 6-AHA is...