The dissociation of apo-and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo-and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0 -3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 3 Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.

Oxamniquine resistance evolved in the human blood fluke (Schistosoma mansoni) in Brazil in the 1970s. We crossed parental parasites differing ~500-fold in drug response, determined drug sensitivity and marker segregation in clonally-derived F2s, and identified a single QTL (LOD=31) on chromosome 6. A sulfotransferase was identified as the causative gene using RNAi knockdown and biochemical complementation assays and we subsequently demonstrated independent origins of loss-of-function mutations in field-derived and laboratory-selected resistant parasites. These results demonstrate the utility of linkage mapping in a human helminth parasite, while crystallographic analyses of protein-drug interactions illuminate the mode of drug action and provide a framework for rational design of oxamniquine derivatives that kill both S. mansoni and S. haematobium, the two species responsible for >99% of schistosomiasis cases worldwide.

Macrophages can be polarized to exhibit either pro-inflammatory M1 or pro-angiogenic M2 phenotypes, but have high phenotypic plasticity. This pilot study investigated macrophage polarization in the macular retina and choroid of age-related macular degeneration (AMD) and non-AMD subjects, as well as in AMD choroidal neovascular membranes (CNVM). All specimens were evaluated for routine histopathology. Quantitative real-time polymerase chain reaction for representative M1 (CXCL11) and M2 (CCL22) transcripts were performed on macular choroidal trephines (MCT) of 19 AMD and nine non-AMD eye bank eyes, on the microdissected macular retinal cells from the archived slides of five geographic atrophic AMD, five exudative/neovascular AMD, and eight normal autopsied eyes, and on microdissected inflammatory cells from two surgically removed CNVM that did not respond to anti-vascular endothelial growth factor (VEGF) therapy. High M2-chemokine transcript and a low ratio of M1 to M2 chemokine transcript were found in aging non-AMD MCT. Advanced AMD maculae had a higher M1 to M2 chemokine transcript ratio compared to normal autopsied eyes. Macrophages in the two CNVM of patients unresponsive to anti-VEGF therapy were polarized toward either M1 or M2 phenotypes. The number of M2 macrophages was increased compared to M1 macrophages in normal aging eyes. A pathological shift of macrophage polarization may play a potential role in AMD pathogenesis.

Kallikrein-4 (KLK4) is a serine proteinase believed to be important in the normal development of dental enamel. We isolated native KLK4 from developing pig enamel and expressed four recombinant forms. Pig KLK4 was expressed in bacteria with and without the propeptide, and in two eukaryotic systems. Recombinant pig KLK4 was secreted as a zymogen by '293' cells and purified. The proKLK4 was activated in vitro by thermolysin and recombinant pig enamelysin, but not by native KLK4. These results were confirmed using a fluorescent peptide analog of the KLK4 propeptide-enzyme junction. Native KLK4 appears as a doublet at 37 kDa and 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Removal of N-linked oligosaccharides by digestion with deglycosidase-F reduced the doublet to a single band at approximately 28 kDa, demonstrating that the active enzyme is glycosylated, and that the 37 kDa and 34 kDa forms differ only in their number of glycosylations. Deglycosylation was also associated with a loss of proteolytic activity. We digested recombinant pig amelogenin with native KLK4 and characterized the cleavage products by N-terminal sequencing and mass spectrometry. Eleven cleavage sites in the amelogenin protein were identified, demonstrating that KLK4 degrades amelogenin and is likely to participate in the degradation of enamel proteins in vivo.

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.. ) is a reactive byproduct of mitochondrial respiration and fatty acid oxidation that can damage critical cellular components. To protect against such damage, cells express antioxidant enzymes such as copper-zinc superoxide dismutase (SOD1) 5 (1), catalase (2), and glutathione peroxidase (3) that together act to convert superoxide to molecular oxygen and water using redox-active metal cofactors. SOD1 comprises between 0.1 and 2.0% of the detergent-soluble protein in spinal cord and brain (4, 5), and this abundance presumably protects against the plentiful superoxide generated by these metabolically active (respiring) tissues. However, expression of SOD1 variants can be harmful if the molecule functions abnormally as demonstrated by dominantly inherited mutations in the human SOD1 gene that give rise to familial forms of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) (6, 7).Seminal studies in transgenic mice have established that pathogenic SOD1 proteins elicit motor neuron dysfunction through the acquisition of a deleterious property and not a loss of enzymatic function (8 -10). Proteinaceous inclusions enriched in SOD1 are observed in cell culture model systems, ALS-SOD1 transgenic mice, and in familial ALS patients, suggesting that SOD1-linked ALS pathology may be related to protein misfolding or aggregation (for reviews, see Refs. 11-13). However, the molecular mechanisms underlying SOD1 toxicity to motor neurons remain unknown, and it remains to be clarified whether the observed inclusions are causal or symptomatic of motor neuron dysfunction.Since the discovery of the SOD1-ALS link nearly 15 years ago, the pathogenic human SOD1 variant in which Arg is substituted for Gly at position 85 (G85R) has been frequently studied. When expressed in transgenic mice, G85R SOD1 causes rapidly progressive motor neuron degeneration while remaining at low levels in the soluble fraction of spinal cord extracts (14 -16). Visible SOD1-containing inclusions first become apparent in astrocytes with the o...

Over 100 mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Two pathogenic SOD1 mutations, His46Arg (H46R) and His48Gln (H48Q), affect residues that act as copper ligands in the wild type enzyme. Transgenic mice expressing a human SOD1 variant containing both mutations develop paralytic disease akin to ALS. Here we show that H46R/H48Q SOD1 possesses multiple characteristics that distinguish it from the wild type. These properties include: 1) an ablated copper-binding site; 2) a substantially weakened affinity for zinc; 3) a binding site for calcium ion; 4) the ability to form stable heterocomplexes with the Copper Chaperone for SOD1 (CCS); and 5) compromised CCS-mediated oxidation of the intrasubunit disulfide bond in vivo. The results presented here, together with data on pathogenic SOD1 proteins coming from cell culture and transgenic mice, suggest that incomplete posttranslational modification of nascent SOD1 polypeptides via CCS may be a characteristic shared by fALS SOD1 mutants, leading to a population of destabilized, off-pathway folding intermediates that are toxic to motor neurons.The homodimeric antioxidant enzyme copper-zinc superoxide dismutase (SOD1) has been studied for nearly four decades. In 1993, interest in the molecule intensified when mutations in the gene encoding SOD1 were linked to the lethal neurodegenerative disease amyotrophic lateral sclerosis (ALS) (1,2). Since then, ∼100 distinct pathogenic mutations have been documented [reviewed in (3)], with most resulting in single amino acid substitutions and a few in truncations in the C-terminal portion of the poypeptide. . Supporting Information Available: Supplementary Figure 1 shows additional details of the calcium binding site and its role in SOD1 crystal packing interactions. Supplementary Figure 2 shows additional details of H46R/H48Q SOD1 interactions with CCS in solution using analytical ultracentrifugation and analytical gel filtration. This material is available free of charge via the Internet at http://pubs.acs.org". NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 April 21. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptLandmark studies in transgenic mice established that pathogenic SOD1 proteins elicit motor neuron dysfunction through the acquisition of a deleterious property and not a loss of enzymatic function (4-6). SOD1-enriched inclusions are observed in cell culture model systems, ALS-SOD1 transgenic mice, and in fALS patients, suggesting that SOD1-linked ALS pathology is related to misfolding or aggregation [reviewed in (7-9)]. However, the precise molecular mechanism(s) underlying SOD1 toxicity to motor neurons is unknown, and it remains to be clarified whether the observed inclusions are causal or symptomatic of motor neuron dysfunction.Given the observations described above, ALS-mutant SOD1 proteins likely possess propert...

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The regulatory protein ToxT is an AraC family protein that is responsible for activating transcription of the genes encoding cholera toxin and toxin coregulated pilus, which are required for virulence by the human pathogen Vibrio cholerae. The N terminus of ToxT contains dimerization and regulatory elements, whereas the C terminus contains the DNA binding domain. Bile and long chain fatty acids negatively regulate ToxT activity. Utilizing a comprehensive alanine substitution mutant library of ToxT, 19 N-terminal residues were found to be critical for dimerization and transcriptional activation. One of these mutant proteins (F151A) was confirmed to be monomeric via centrifugation and exhibited a weakened ability to bind to the tcpA promoter in a gel mobility shift assay. Moreover, a V. cholerae toxTF151A mutant failed to colonize the infant mouse intestine, emphasizing the importance of ToxT N-terminal dimerization to cholera pathogenesis. Six N-terminal alanine substitutions allowed ToxT transcriptional activity in the presence of inhibitory concentrations of bile, palmitoleic acid, and the small molecule inhibitor virstatin. Two of these mutations (N106A and L114A) enhance N-terminal dimerization in a bacterial twohybrid system reconstituted in V. cholerae, which is otherwise disrupted by bile, palmitoleic acid, and virstatin. We demonstrate that V. cholerae toxTN106A and toxTL114A strains colonize the infant mouse intestine at significantly higher levels than the wild type strain. Our results demonstrate that ToxT N-terminal dimerization is required for transcriptional activation and cholera pathogenesis and that fatty acids modulate ToxT activity via modulation of dimerization.Vibrio cholerae causes the disease cholera, a life-threatening diarrheal illness that affects thousands of people annually (1). The bacterium is acquired through the consumption of contaminated food or water and colonizes the small intestine (2). In the intestinal environment, V. cholerae expresses two critical virulence factors that facilitate colonization and disease symptoms, cholera toxin (CT) 2 and toxin-coregulated pilus (TCP). CT is an ADP-ribosylating toxin that is translocated into host cells and modifies G s ␣, causing an ion imbalance that leads to the profuse diarrhea associated with the disease (2). TCP is a type IV bundle-forming pilus that is required for intestinal colonization (3).Expression of CT and TCP is coordinately regulated by the transcriptional activator ToxT (4). ToxT, an AraC/XylS family activator, directly binds to the promoters of the ctx and tcp genes (which encode CT and TCP) and activates their transcription (5). V. cholerae strains lacking ToxT express no CT or TCP and are unable to colonize the intestine and cause disease, emphasizing the central role this regulatory protein plays in cholera pathogenesis (6). Transcription of toxT is regulated by a virulence cascade commonly referred to as the ToxR regulon, which responds to various environmental stimuli to ensure that ToxT is only expressed within the...