Solution structure of a monoheme ferrocytochrome c from Shewanella putrefaciens and structural analysis of sequence-similar proteins: functional implications

Within the frame of the characterization of the structure and function of cytochromes c, an 81-amino acid cytochrome c was identified in the genome of Shewanella putrefaciens. Because of the scarce information about bacterial cytochromes of this type and the large variability in sequences and possibly function, we decided to proceed to its structural characterization. This protein was expressed in Escherichia coli and purified. The oxidized species is largely high spin, with a detached methionine, whereas the reduced species has the classical His/Met axial ligation to iron. The NMR solution structure of the reduced form was determined on a (15)N-labeled sample, for which 99% of all non-proline backbone (1)H and (15)N resonances have been assigned. One thousand three hundred two meaningful NOEs, out of 1775 NOEs, together with 66 dihedral angles provide a structure with rmsd values from the mean of 0.50 and 0.96 A for backbone and all heavy atoms, respectively. A search of gene banks allowed us to locate 10 different cytochromes c, the sequences of which are more than 30% identical to that of the S. putrefacienscytochrome. For two of them, the structures are known. The structures of the others have been modeled by using the available templates and internally validated. Structural similarities in terms of surface properties account for their biophysical features and provide hints about the function.     

A further clue to understanding the mobility of mitochondrial yeast cytochrome c: a (15)N T1rho investigation of the oxidized and reduced species.

A new approach was developed to overproduce 15N-enriched yeast iso-1-cytochrome c in the periplasm of Escherichia coli in order to perform a study of the motions in the ms-micros time scale on the oxidized and reduced forms through rotating frame 15N relaxation rates and proton/deuterium exchange studies. It is confirmed that the reduced protein is rather rigid whereas the oxidized species is more flexible. The regions of the protein that display increased internal mobility upon oxidation are easily identified by the number of residues experiencing conformational equilibria and by their exchange rates. These data complement the information already available in the literature and provide a comprehensive picture of the mobility in the protein. In particular, oxidation mobilizes the loop containing Met80 and, through specific contacts, affects the mobility of helix 3 and possibly of helix 5, and of a section of protein connecting the heme propionates to helix 2. The relevance of internal motions to molecular recognition and to the early steps of the unfolding process of the oxidized species is also discussed. In agreement with the reported data, subnanosecond mobility is found to be less informative than the ms-micros with respect to redox dependent properties.     

Protein quality control during aging involves recruitment of the macroautophagy pathway by BAG3

The Hsc/Hsp70 co-chaperones of the BAG (Bcl-2-associated athanogene) protein family are modulators of protein quality control. We examined the specific roles of BAG1 and BAG3 in protein degradation during the aging process. We show that BAG1 and BAG3 regulate proteasomal and macroautophagic pathways, respectively, for the degradation of polyubiquitinated proteins. Moreover, using models of cellular aging, we find that a switch from BAG1 to BAG3 determines that aged cells use more intensively the macroautophagic system for turnover of polyubiquitinated proteins. This increased macroautophagic flux is regulated by BAG3 in concert with the ubiquitin-binding protein p62/SQSTM1. The BAG3/BAG1 ratio is also elevated in neurons during aging of the rodent brain, where, consistent with a higher macroautophagy activity, we find increased levels of the autophagosomal marker LC3-II as well as a higher cathepsin activity. We conclude that the BAG3-mediated recruitment of the macroautophagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro-oxidant and aggregation-prone milieu characteristic of aging.     

Novel imine antioxidants at low nanomolar concentrations protect dopaminergic cells from oxidative neurotoxicity

Strong evidence indicates that oxidative stress may be causally involved in the pathogenesis of Parkinson's disease. We have employed human dopaminergic neuroblastoma cells and rat primary mesencephalic neurons to assess the protective potential of three novel bisarylimine antioxidants on dopaminergic cell death induced by complex I inhibition or glutathione depletion. We have found that exceptionally low concentrations (EC₅₀ values ∼20 nM) of these compounds (iminostilbene, phenothiazine, and phenoxazine) exhibited strong protective effects against the toxicities of MPP⁺, rotenone, and l -buthionine sulfoximine. Investigating intracellular glutathione levels, it was found that MPP⁺, l -buthionine sulfoximine, and rotenone disrupted different aspects of the native glutathione equilibrium, while the aromatic imines did not further influence glutathione levels or redox state on any baseline. However, the imines independently reduced protein oxidation and total oxidant flux, saved the mitochondrial membrane potential, and provided full cytoprotection under conditions of complete glutathione depletion. The unusually potent antioxidant effects of the bisarylimines could be reproduced in isolated mitochondria, which were instantly protected from lipid peroxidation and pathological swelling. Aromatic imines may be interesting lead structures for a potential antioxidant therapy of Parkinson's disease and other disorders accompanied by glutathione dysregulation.     

NXT2 is required for embryonic heart development in zebrafish

Background  

NXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes.     

Membrane protein oxidation determines neuronal degeneration

Oxidative stress is an early hallmark in neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. However, the critical biochemical effector mechanisms of oxidative neurotoxicity have remained surprisingly elusive. In screening various peroxides and potential substrates of oxidation for their effect on neuronal survival, we observed that intramembrane compounds were significantly more active than aqueous or amphiphilic compounds. To better understand this result, we synthesized a series of competitive and site‐specific membrane protein oxidation inhibitors termed aminoacyllipids, whose structures were designed on the basis of amino acids frequently found at the protein‐lipid interface of synaptic membrane proteins. Investigating the aminoacyllipids in primary neuronal culture, we found that the targeted protection of transmembrane tyrosine and tryptophan residues was sufficient to prevent neurotoxicity evoked by hydroperoxides, kainic acid, glutathione‐depleting drugs, and certain amyloidogenic peptides, but ineffective against non‐oxidative inducers of apoptosis such as sphingosine or Akt kinase inhibitors. Thus, the oxidative component of different neurotoxins appears to converge on neuronal membrane proteins, irrespective of the primary mechanism of cellular oxidant generation. Our results indicate the existence of a one‐electron redox cycle based on membrane protein aromatic surface amino acids, whose disturbance or overload leads to excessive membrane protein oxidation and neuronal death.

     

812. IRIS SCHELKOWNIKOWII

Iris schelkownikowii (Fomin) Fomin, a little‐known species from Azerbaijan in section Oncocyclus, is described and illustrated. Suggestions for its cultivation are given.     

Dimethyl propionate ester heme-containing cytochrome b5: structure and stability.

A derivative of rat microsomal cytochrome b5, obtained by substitution of the native heme moiety with protoporphyrin IX dimethyl ester, has been characterized by 1H and 15N NMR spectroscopy. Besides the two usual A and B forms, which depend on the orientation of the heme in the prostethic group cavity, two other minor forms have been detected which presumably indicate different conformations of the vinyl side chains. The shifts of the heme methyls, as well as the directions of the rhombic axes of the magnetic susceptibility tensor, indicate a small difference in the orientation of the imidazole planes of the histidine axial ligands. The solution structure was determined by using 1,303 meaningful NOEs and 241 pseudocontact shifts, the latter being derived from the native reduced protein. A family of 40 energy-minimized conformers was obtained with average RMSD of 0.56+/-0.09 A and 1.04+/-0.12 A for backbone and heavy atoms, respectively, and distance and pseudocontact shift penalty functions of 0.50+/-0.07 A2 and 0.51+/-0.02 ppm2. The structure shows some changes around the cavity and in particular a movement of the 60-70 backbone segment owing to the absence of two hydrogen bonds between the Ser64 backbone NH and side-chain OH and the carboxylate oxygen of propionate-7, present in the native protein. The analysis of the NMR spectra in the presence of unfolding agents indicates that this protein is less stable than the native form. The decrease in stability may be the result of the loss of the two hydrogen bonds connecting propionate-7 to Ser64 in the native protein. The available data on the reduction potential and the electron transfer rates are discussed on the basis of the present structural data.     

Side chain mobility as monitored by CH-CH cross correlation: The example of cytochrome b5

The mobility of CH₂ moieties in oxidized and reduced cytochrome b ₅ was studied by analyzing the ¹³C relaxation of the J-split components, in terms of C-H dipole–C-H dipole cross correlation rates. A 2D ¹³C-¹H experiment is proposed to measure these rates that provide the internal effective reorientation correlation time for each CH₂ moiety. It is found that higher mobility is present in the helices forming the heme pocket. On the contrary, the strands, which form the hydrophobic core of the molecule, have the lowest mobility. The general pattern is the same for the oxidized and reduced species, indicating that any oxidation-dependent property detected for backbone NH moieties does not affect the CH₂ mobility.