Role of ALDP (ABCD1) and mitochondria in X-linked adrenoleukodystrophy

Peroxisomal disorders have been associated with malfunction of peroxisomal metabolic pathways, but the pathogenesis of these disorders is largely unknown. X-linked adrenoleukodystrophy (X-ALD) is associated with elevated levels of very-long-chain fatty acids (VLCFA; C(>22:0)) that have been attributed to reduced peroxisomal VLCFA beta-oxidation activity. Previously, our laboratory and others have reported elevated VLCFA levels and reduced peroxisomal VLCFA beta-oxidation in human and mouse X-ALD fibroblasts. In this study, we found normal levels of peroxisomal VLCFA beta-oxidation in tissues from ALD mice with elevated VLCFA levels. Treatment of ALD mice with pharmacological agents resulted in decreased VLCFA levels without a change in VLCFA beta-oxidation activity. These data indicate that ALDP does not determine the rate of VLCFA beta-oxidation and that VLCFA levels are not determined by the rate of VLCFA beta-oxidation. The rate of peroxisomal VLCFA beta-oxidation in human and mouse fibroblasts in vitro is affected by the rate of mitochondrial long-chain fatty acid beta-oxidation. We hypothesize that ALDP facilitates the interaction between peroxisomes and mitochondria, resulting, when ALDP is deficient in X-ALD, in increased VLCFA accumulation despite normal peroxisomal VLCFA beta-oxidation in ALD mouse tissues. In support of this hypothesis, mitochondrial structural abnormalities were observed in adrenal cortical cells of ALD mice.     

NMR structure of free RGS4 reveals an induced conformational change upon binding Galpha

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) are transducers in many cellular transmembrane signaling systems where regulators of G-protein signaling (RGS) act as attenuators of the G-protein signal cascade by binding to the Galpha subunit of G-proteins (G(i)(alpha)(1)) and increasing the rate of GTP hydrolysis. The high-resolution solution structure of free RGS4 has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geometry-simulated annealing using a total of 2871 experimental NMR restraints. The atomic rms distribution about the mean coordinate positions for residues 5-134 for the 30 structures is 0.47 +/- 0.05 A for the backbone atoms, 0. 86 +/- 0.05 A for all atoms, and 0.56 +/- 0.04 A for all atoms excluding disordered side chains. The NMR solution structure of free RGS4 suggests a significant conformational change upon binding G(i)(alpha)(1) as evident by the backbone atomic rms difference of 1. 94 A between the free and bound forms of RGS4. The underlying cause of this structural change is a perturbation in the secondary structure elements in the vicinity of the G(i)(alpha)(1) binding site. A kink in the helix between residues K116-Y119 is more pronounced in the RGS4-G(i)(alpha)(1) X-ray structure relative to the free RGS4 NMR structure, resulting in a reorganization of the packing of the N-terminal and C-terminal helices. The presence of the helical disruption in the RGS4-G(i)(alpha)(1) X-ray structure allows for the formation of a hydrogen-bonding network within the binding pocket for G(i)(alpha)(1) on RGS4, where RGS4 residues D117, S118, and R121 interact with residue T182 from G(i)(alpha)(1). The binding pocket for G(i)(alpha)(1) on RGS4 is larger and more accessible in the free RGS4 NMR structure and does not present the preformed binding site observed in the RGS4-G(i)(alpha)(1) X-ray structure. This observation implies that the successful complex formation between RGS4 and G(i)(alpha)(1) is dependent on both the formation of the bound RGS4 conformation and the proper orientation of T182 from G(i)(alpha)(1). The observed changes for the free RGS4 NMR structure suggest a mechanism for its selectivity for the Galpha-GTP-Mg(2+) complex and a means to facilitate the GTPase cycle.     

Analysis by NMR spectroscopy of the structural homology between the linear and the cyclic peptide recognized by anti-human leukocyte antigen class I monoclonal antibody TP25.99*

The anti-human leukocyte antigen (HLA) class I monoclonal antibody (mAb) TP25.99 has a unique specificity since it recognizes both a conformational and a linear determinant expressed on the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains, respectively. Previously, we reported the identification of a cyclic and a linear peptide that inhibits mAb TP25.99 binding to the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains (S. A. Desai, X. Wang, E. J. Noronha, Q. Zhou, V. Rebmann, H. Grosse-Wilde, F. J. Moy, R. Powers, and S. Ferrone, submitted for publication). The linear X(19) and cyclic LX-8 peptides contain sequence homologous to residues 239-242, 245, and 246 and to residues 194-198, respectively, of HLA class I heavy chain alpha(3) domain. Analysis by two-dimensional transfer nuclear Overhauser effect spectroscopy of the induced solution structures of the linear X(19) and cyclic LX-8 peptides in the presence of mAb TP25.99 showed that the two peptides adopt a similar structural motif despite the lack of sequence homology. The backbone fold is suggestive of a short helical segment followed by a tight turn, reminiscent of the determinant loop region (residues 194-198) on beta(2)-mu-associated HLA class I heavy chains. The structural similarity between the linear X(19) and cyclic LX-8 peptides and the lack of sequence homology suggests that mAb TP25.99 predominantly recognizes a structural motif instead of a consensus sequence.     

A functional pseudoknot in 16S ribosomal RNA.

Several lines of evidence indicate that the universally conserved 530 loop of 16S ribosomal RNA plays a crucial role in translation, related to the binding of tRNA to the ribosomal A site. Based upon limited phylogenetic sequence variation, Woese and Gutell (1989) have proposed that residues 524-526 in the 530 hairpin loop are base paired with residues 505-507 in an adjoining bulge loop, suggesting that this region of 16S rRNA folds into a pseudoknot structure. Here, we demonstrate that Watson-Crick interactions between these nucleotides are essential for ribosomal function. Moreover, we find that certain mild perturbations of the structure, for example, creation of G-U wobble pairs, generate resistance to streptomycin, an antibiotic known to interfere with the decoding process. Chemical probing of mutant ribosomes from streptomycin-resistant cells shows that the mutant ribosomes have a reduced affinity for streptomycin, even though streptomycin is thought to interact with a site on the 30S subunit that is distinct from the 530 region. Data from earlier in vitro assembly studies suggest that the pseudoknot structure is stabilized by ribosomal protein S12, mutations in which have long been known to confer streptomycin resistance and dependence.     

Purification and cloning of a novel serine protease, RNK-Met-1, from the granules of a rat natural killer cell leukemia.

We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.     

Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA.

Complexes between 16S rRNA and purified ribosomal proteins, either singly or in combination, were assembled in vitro and probed with hydroxyl radicals generated from free Fe(II)-EDTA. The broad specificity of hydroxyl radicals for attack at the ribose moiety in both single- and double-stranded contexts permitted probing of nearly all of the nucleotides in the 16S rRNA chain. Specific protection of localized regions of the RNA was observed in response to assembly of most of the ribosomal proteins. The locations of the protected regions were in good general agreement with the footprints previously reported for base-specific chemical probes, and with sites of RNA-protein crosslinking. New information was obtained about interaction of ribosomal proteins with 16S rRNA, especially with helical elements of the RNA. In some cases, 5' or 3' stagger in the protection pattern on complementary strands suggests interaction of proteins with the major or minor groove, respectively, of the RNA. These results reinforce and extend previous data on the localization of ribosomal proteins with respect to structural features of 16S rRNA, and offer many new constraints for three-dimensional modeling of the 30S ribosomal subunit.     

Improved cardiac performance after ischemia in aged rats supplemented with vitamin E and alpha-lipoic acid

The purpose of these experiments was to examine the effects of dietary antioxidant supplementation with vitamin E (VE) and alpha-lipoic acid (alpha-LA) on biochemical and physiological responses to in vivo myocardial ischemia-reperfusion (I-R) in aged rats. Male Fischer-334 rats (18 mo old) were assigned to either 1) a control diet (CON) or 2) a VE and alpha-LA supplemented diet (ANTIOX). After a 14-wk feeding period, animals in each group underwent an in vivo I-R protocol (25 min of myocardial ischemia and 15 min of reperfusion). During reperfusion, peak arterial pressure was significantly higher (P < 0.05) in ANTIOX animals compared with CON diet animals. I-R resulted in a significant increase (P < 0.05) in myocardial lipid peroxidation in CON diet animals but not in ANTIOX animals. Compared with ANTIOX animals, heart homogenates from CON animals experienced significantly less (P < 0.05) oxidative damage when exposed to five different in vitro radical producing systems. These data indicate that dietary supplementation with VE and alpha-LA protects the aged rat heart from I-R-induced lipid peroxidation by scavenging numerous reactive oxygen species. Importantly, this protection is associated with improved cardiac performance during reperfusion.     

Multivariate data analysis of growth medium trends affecting antibody glycosylation

Use of multivariate data analysis for the manufacturing of biologics has been increasing due to more widespread use of data-generating process analytical technologies (PAT) promoted by the US FDA. To generate a large dataset on which to apply these principles, we used an in-house model CHO DG44 cell line cultured in automated micro bioreactors alongside PAT with four commercial growth media focusing on antibody quality through N-glycosylation profiles. Using univariate analyses, we determined that different media resulted in diverse amounts of terminal galactosylation, high mannose glycoforms, and aglycosylation. Due to the amount of in-process data generated by PAT instrumentation, multivariate data analysis was necessary to ascertain which variables best modeled our glycan profile findings. Our principal component analysis revealed components that represent the development of glycoforms into terminally galacotosylated forms (G1F and G2F), and another that encompasses maturation out of high mannose glycoforms. The partial least squares model additionally incorporated metabolic values to link these processes to glycan outcomes, especially involving the consumption of glutamine. Overall, these approaches indicated a tradeoff between cellular productivity and product quality in terms of the glycosylation. This work illustrates the use of multivariate analytical approaches that can be applied to complex bioprocessing problems for identifying potential solutions.     

ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization

Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects. Characterizations of beta-barrel (H43R) and dimer interface (A4V) FALS mutants reveal reduced stability and drastically increased aggregation propensity. Moreover, electron and atomic force microscopy indicate that these defects promote the formation of filamentous aggregates. The filaments resemble those seen in neurons of FALS patients and bind both Congo red and thioflavin T, suggesting the presence of amyloid-like, stacked beta-sheet interactions. These results support free-cysteine-independent aggregation of FALS mutant SOD as an integral part of FALS pathology. They furthermore provide a molecular basis for the single FALS disease phenotype resulting from mutations of diverse side-chains throughout the protein: many FALS mutations reduce structural integrity, lowering the energy barrier for fibrous aggregation.