Nitric oxide (NO) is a key physiological mediator and disturbed regulation of NO release is associated with the pathophysiology of almost all inflammatory diseases. A multitude of inhibitors of NOSs (nitric oxide synthases) have been developed, initially with low or even no selectivity against the constitutively expressed NOS isoforms, eNOS (endothelial NOS) and nNOS (neuronal NOS). In the meanwhile these efforts yielded potent and highly selective iNOS (inducible NOS) inhibitors. Moreover, iNOS inhibitors have been shown to exert beneficial anti-inflammatory effects in a wide variety of acute and chronic animal models of inflammation. In the present mini-review, we summarize some of our current knowledge of inhibitors of the iNOS isoenzyme, their biochemical properties and efficacy in animal models of pulmonary diseases and in human disease itself. Moreover, the potential benefit of iNOS inhibition in animal models of COPD (chronic obstructive pulmonary disease), such as cigarette smoke-induced pulmonary inflammation, has not been explicitly studied so far. In this context, we demonstrated recently that both a semi-selective iNOS inhibitor {l-NIL [N6-(1-iminoethyl)-l-lysine hydrochloride]} and highly selective iNOS inhibitors (GW274150 and BYK402750) potently diminished inflammation in a cigarette smoke mouse model mimicking certain aspects of human COPD. Therefore, despite the disappointing results from recent asthma trials, iNOS inhibition could still be of therapeutic utility in COPD, a concept which needs to be challenged and validated in human disease.

The mitochondrial outer membrane contains two distinct machineries for protein import and protein sorting that function in a sequential manner: the general translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex), which is dedicated to ␤-barrel proteins. The SAM core complex consists of three subunits, Sam35, Sam37, and Sam50, that can associate with a fourth subunit, the morphology component Mdm10, to form the SAM holo complex. Whereas the SAM core complex is required for the biogenesis of all ␤-barrel proteins, Mdm10 and the SAM holo complex play a selective role in ␤-barrel biogenesis by promoting assembly of Tom40 but not of porin. We report that Tom7, a conserved subunit of the TOM complex, functions in an antagonistic manner to Mdm10 in biogenesis of Tom40 and porin. We show that Tom7 promotes segregation of Mdm10 from the SAM holo complex into a low molecular mass form. Upon deletion of Tom7, the fraction of Mdm10 in the SAM holo complex is significantly increased, explaining the opposing functions of Tom7 and Mdm10 in ␤-barrel sorting. Thus the role of Tom7 is not limited to the TOM complex. Tom7 functions in mitochondrial protein biogenesis by a new mechanism, segregation of a sorting component, leading to a differentiation of ␤-barrel assembly.Mitochondria import the vast majority of their ϳ1,000 different proteins from the cytosol (1-3). Upon initial recognition and translocation by the general translocase of the outer mitochondrial membrane (TOM 5 complex), different classes of precursor proteins are distributed to the four mitochondrial subcompartments, outer membrane, intermembrane space, inner membrane, and matrix (4 -11). Preproteins carrying cleavable amino-terminal targeting signals (presequences) are transferred from the TOM complex to the presequence translocase of the inner membrane (TIM23 complex) and then sorted to matrix and inner membrane. Many mitochondrial proteins, however, are synthesized without cleavable presequences, including all outer membrane proteins and the majority of intermembrane space and inner membrane proteins. These proteins use distinct import and sorting machineries. Thus, in addition to the presequence pathway, three sorting pathways for non-cleavable mitochondrial precursor proteins have been identified, each of them starting at the TOM complex. Many hydrophobic inner membrane proteins, including the abundant metabolite carriers, are imported via small Tim proteins of the intermembrane space and the twin pore translocase of the inner membrane (TIM22 complex) (8, 12). Small proteins of the intermembrane space use a special import and assembly machinery that includes Mia40 and the sulfhydryl oxidase Erv1 (13-17). Outer membrane proteins are also initially transported via the TOM complex; however, the TOM complex is not able to integrate ␤-barrel proteins into the membrane. Therefore, the outer membrane contains a separate sorting and assembly machinery (SAM complex) that directs membrane integration and assembly of ␤-barrel...

Ssc1, a molecular chaperone of the Hsp70 family, drives preprotein import into the mitochondrial matrix by a speci®c interaction with the translocase component Tim44. Two other mitochondrial Hsp70s, Ssc3 (Ecm10) and Ssq1, show high sequence homology to Ssc1 but fail to replace Ssc1 in vivo, possibly due to their inability to interact with Tim44. We analyzed the structural basis of the Tim44 interaction by the construction of chimeric Hsp70 proteins. The ATPase domains of all three mitochondrial Hsp70s were shown to bind to Tim44, supporting the active motor model for the Hsp70 mechanism during preprotein translocation. The peptide-binding domain of Ssc1 sustained binding of Tim44, while the peptide-binding domains of Ssc3 and Ssq1 exerted a negative effect on the interaction of the ATPase domains with Tim44. A mutation in the peptide-binding domain of Ssc1 resulted in a similar negative effect not only on the ATPase domain of Ssc1, but also of Ssq1 and Ssc3. Hence, the determination of a crucial Hsp70 function via the peptide-binding domain suggests a new regulatory principle for Hsp70 domain cooperation.

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Mitochondrial proteins are synthesized as precursor proteins in the cytosol and are posttranslationally imported into the organelle. A complex system of translocation machineries recognizes and transports the precursor polypeptide across the mitochondrial membranes. Energy for the translocation process is mainly supplied by the mitochondrial membrane potential (⌬) and the hydrolysis of ATP. Mitochondrial Hsp70 (mtHsp70) has been identified as the major ATPase driving the membrane transport of the precursor polypeptides into the mitochondrial matrix. Together with the partner proteins Tim44 and Mge1, mtHsp70 forms an import motor complex interacting with the incoming preproteins at the inner face of the inner membrane. This import motor complex drives the movement of the polypeptides in the translocation channel and the unfolding of carboxy-terminal parts of the preproteins on the outside of the outer membrane. Two models of the molecular mechanism of mtHsp70 during polypeptide translocation are discussed. In the 'trapping' model, precursor movement is generated by Brownian movement of the polypeptide chain in the translocation pore. This random movement is made vectorial by the interaction with mtHsp70 in the matrix. The detailed characterization of conditional mutants of the import motor complex provides the basis for an extended model. In this 'pulling' model, the attachment of mtHsp70 at the inner membrane via Tim44 and a conformational change induced by ATP results in the generation of an inward-directed force on the bound precursor polypeptide. This active role of the import motor complex is necessary for the translocation of proteins containing tightly folded domains. We suggest that both mechanisms complement each other to reach a high efficiency of preprotein import.