Neuropathogenic forms of huntingtin and androgen receptor inhibit fast axonal transport

Huntington's and Kennedy's disease are autosomal dominant neurodegenerative diseases caused by pathogenic expansion of polyglutamine tracts. Expansion of glutamine repeats must in some way confer a gain of pathological function that disrupts an essential cellular process and leads to loss of affected neurons. Association of huntingtin with vesicular structures raised the possibility that axonal transport might be altered. Here we show that polypeptides containing expanded polyglutamine tracts, but not normal N-terminal huntingtin or androgen receptor, directly inhibit both fast axonal transport in isolated axoplasm and elongation of neuritic processes in intact cells. Effects were greater with truncated polypeptides and occurred without detectable morphological aggregates.     

NF-kappa B inhibition by omega -3 fatty acids modulates LPS-stimulated macrophage TNF-alpha transcription

Omega-3 fatty acid (FA) emulsions reduce LPS-stimulated murine macrophage TNF-alpha production, but the exact mechanism has yet to be defined. The purpose of this study was to determine the mechanism for omega-3 FA inhibition of macrophage TNF-alpha production following LPS stimulation. RAW 264.7 cells were pretreated with isocaloric emulsions of omega-3 FA (Omegaven), omega-6 FA (Lipovenos), or DMEM and subsequently exposed to LPS. IkappaB-alpha and phospho-IkappaB-alpha were determined by Western blotting. NF-kappaB binding was assessed using the electromobility shift assay, and activity was measured using a luciferase reporter vector. RT-PCR and ELISA quantified TNF-alpha mRNA and protein levels, respectively. Pretreatment with omega-3 FA inhibited IkappaB phosphorylation and significantly decreased NF-kappaB activity. Moreover, omega-3-treated cells demonstrated significant decreases in both TNF-alpha mRNA and protein expression by 47 and 46%, respectively. These experiments demonstrate that a mechanism for proinflammatory cytokine inhibition in murine macrophages by omega-3 FA is mediated, in part, through inactivation of the NF-kappaB signal transduction pathway secondary to inhibition of IkappaB phosphorylation.     

Structure of tetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase

How 6R-tetrahydrobiopterin (H(4)B) participates in Arg hydroxylation as catalyzed by the nitric oxide synthases (NOSs) is a topic of current interest. Previous work with the oxygenase domain of inducible NOS (iNOSoxy) demonstrated that H(4)B radical formation is kinetically coupled to disappearance of an initial heme-dioxy intermediate and to Arg hydroxylation in a single turnover reaction run at 10 degrees C [Wei, C.-C., Wang, Z.-Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) J. Biol. Chem. 276, 315-319]. Here we used 5-methyl-H(4)B to investigate how pterin structure influences radical formation and associated catalytic steps. In the presence of Arg, the heme-dioxy intermediate in 5-methyl-H(4)B-bound iNOSoxy reacted at a rate of 35 s(-)(1), which is 3-fold faster than with H(4)B. This was coupled to a faster rate of 5-methyl-H(4)B radical formation (40 vs 12.5 s(-)(1)) and to a faster and more productive Arg hydroxylation. The EPR spectrum of the enzyme-bound 5-methyl-H(4)B radical had different hyperfine structure than the bound H(4)B radical and exhibited a 3-fold longer half-life after its formation. A crystal structure of 5-methyl-H(4)B-bound iNOSoxy revealed that there are minimal changes in conformation of the bound pterin or in its interactions with the protein as compared to H(4)B. Together, we conclude the following: (1) The rate of heme-dioxy reduction is linked to pterin radical formation and is sensitive to pterin structure. (2) Faster heme-dioxy reduction increases the efficiency of Arg hydroxylation but still remains rate limiting for the reaction. (3) The 5-methyl group influences heme-dioxy reduction by altering the electronic properties of the pterin rather than changing protein structure or interactions. (4) Faster electron transfer from 5-methyl-H(4)B may be due to increased radical stability afforded by the N-5 methyl group.     

The GxxxG motif: a framework for transmembrane helix-helix association

In order to identify strong transmembrane helix packing motifs, we have selected transmembrane domains exhibiting high-affinity homo-oligomerization from a randomized sequence library based on the right-handed dimerization motif of glycophorin A. Sequences were isolated using the TOXCAT system, which measures transmembrane helix-helix association in the Escherichia coli inner membrane. Strong selection was applied to a large range of sequences ( approximately 10(7) possibilities) and resulted in the identification of sequence patterns that mediate high-affinity helix-helix association. The most frequent motif isolated, GxxxG, occurs in over 80% of the isolates. Additional correlations suggest that flanking residues act in concert with the GxxxG motif, and that size complementarity is maintained at the interface, consistent with the idea that the identified sequence patterns represent packing motifs. The convergent identification of similar sequence patterns from an analysis of the transmembrane domains in the SwissProt sequence database suggests that these packing motifs are frequently utilized in naturally occurring helical membrane proteins.     

Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1

The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.     

Identification of a Mn-O-Mn cluster vibrational mode of the oxygen-evolving complex in photosystem II by low-frequency FTIR spectroscopy

We have developed conditions for recording the low-frequency S(2)/S(1) Fourier transform infrared difference spectrum of hydrated PSII samples. By exchanging PSII samples with buffered (18)O water, we found that a positive band at 606 cm(-)(1) in the S(2)/S(1) spectrum in (16)O water is clearly downshifted to 596 cm(-)(1) in (18)O water. By taking double-difference (S(2)/S(1) and (16)O minus (18)O) spectra, we assign the 606 cm(-)(1) mode to an S(2) mode and also identify a corresponding S(1) mode at about 625 cm(-)(1). In addition, by Sr and (44)Ca substitution experiments, we found that the 606 cm(-)(1) mode is upshifted to about 618 cm(-)(1) by Sr(2+) substitution but that this mode is not affected by substitution with the (44)Ca isotope. On the basis of these results and also on the basis of studies of Mn model compounds, we assign the 625 cm(-)(1) mode in the S(1) state and the 606 cm(-)(1) mode in the S(2) state to a Mn-O-Mn cluster vibration of the oxygen-evolving complex (OEC) in PSII. This structure may include additional bridge(s), which could be another oxo, carboxylato(s), or atoms derived from an amino acid side chain. Our results indicate that the bridged oxygen atom shown in this Mn-O-Mn cluster is exchangeable and accessible by water. The downshift in the Mn-O-Mn cluster vibration as manganese is oxidized during the S(1) --> S(2) transition is counterintuitive; we discuss possible origins of this behavior. Our results also indicate that Sr(2+) substitution in PSII causes a small structural perturbation that affects the bond strength of the Mn-O-Mn cluster in the PSII OEC. This suggests that Sr(2+), and by inference, Ca(2+), communicates with, but is not integral to, the manganese core.     

D1-Asp170 is structurally coupled to the oxygen evolving complex in photosystem II as revealed by light-induced Fourier transform infrared difference spectroscopy

We report both mid-frequency (1800-1200 cm(-)(1)) and low-frequency (670-350 cm(-)(1)) S(2)/S(1) FTIR difference spectra of photosystem II (PSII) particles isolated from wild-type and D1-D170H mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. Both mid- and low-frequency S(2)/S(1) spectra of the Synechocystis wild-type PSII particles closely resemble those from spinach PSII samples, which confirms an earlier result by Noguchi and co-workers [Noguchi, T., Inoue, Y., and Tang, X.-S. (1997) Biochemistry 36, 14705-14711] and indicates that the coordination environment of the oxygen evolving complex (OEC) in Synechocystis is very similar to that in spinach. We also found that there is no appreciable difference between the mid-frequency S(2)/S(1) spectra of wild-type and of D1-D170H mutant PSII particles, from which we conclude that D1-Asp170 does not undergo a significant structural change during the S(1) to S(2) transition. This result also suggests that, if D1-Asp170 ligates Mn, it does not ligate the Mn ion that is oxidized during the S(1) to S(2) state transition. Finally, we found that a mode at 606 cm(-)(1) in the low-frequency wild-type S(2)/S(1) spectrum shifts to 612 cm(-)(1) in the D1-D170H mutant spectrum. Because this 606 cm(-)(1) mode has been previously assigned to an Mn-O-Mn cluster mode of the OEC [Chu, H.-A., Sackett, H., and Babcock, G. T. (2000) Biochemistry 39, 14371-14376], we conclude that D1-Asp170 is structurally coupled to the Mn-O-Mn cluster structure that gives rise to this band. Our results suggest that D1-Asp170 either directly ligates Mn or Ca(2+) or participates in a hydrogen bond to the Mn(4)Ca(2+) cluster. Our results demonstrate that combining FTIR difference spectroscopy with site-directed mutagenesis has the potential to provide insights into structural changes in Mn and Ca(2+) coordination environments in the different S states of the OEC.