Aggregation-triggering segments of SOD1 fibril formation support a common pathway for familial and sporadic ALS

ALS is a terminal disease of motor neurons that is characterized by accumulation of proteinaceous deposits in affected cells. Pathological deposition of mutated Cu/Zn superoxide dismutase (SOD1) accounts for ∼20% of the familial ALS (fALS) cases. However, understanding the molecular link between mutation and disease has been difficult, given that more than 140 different SOD1 mutants have been observed in fALS patients. In addition, the molecular origin of sporadic ALS (sALS) is unclear. By dissecting the amino acid sequence of SOD1, we identified four short segments with a high propensity for amyloid fibril formation. We find that fALS mutations in these segments do not reduce their propensity to form fibrils. The atomic structures of two fibril-forming segments from the C terminus, ^(101)DSVISLS^(107) and ^(147)GVIGIAQ^(153), reveal tightly packed β-sheets with steric zipper interfaces characteristic of the amyloid state. Based on these structures, we conclude that both C-terminal segments are likely to form aggregates if available for interaction. Proline substitutions in 101DSVISLS107 and ^(147)GVIGIAQ^(153) impaired nucleation and fibril growth of full-length protein, confirming that these segments participate in aggregate formation. Our hypothesis is that improper protein maturation and incompletely folded states that render these aggregation-prone segments available for interaction offer a common molecular pathway for sALS and fALS

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating Cytotoxin from Mycoplasma Pneumoniae

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and walking pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem beta-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal beta-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases

Purification and crystallization of human Cu/Zn superoxide dismutase recombinantly produced in the protozoan Leishmania tarentolae

The rapid and inexpensive production of high-quality eukaryotic proteins in recombinant form still remains a challenge in structural biology. Here, a protein-expression system based on the protozoan Leishmania tarentolae was used to produce human Cu/Zn superoxide dismutase (SOD1) in recombinant form. Sequential integration of the SOD1 expression cassettes was demonstrated to lead to a linear increase in expression levels to up to 30 mg per litre. Chromatographic purification resulted in 90% pure recombinant protein, with a final yield of 6.5 mg per litre of culture. The protein was crystallized and the structures of two new crystal forms were determined. These results demonstrate the suitability of the L. tarentolae expression system for structural research

EFFECTS OF UNCONVENTIONAL PLANT OILS AND RUMEN ADAPTATION ON METHANE GAS EMISSION AND RUMEN FERMENTATION CHARACTERISTICS

The objectives of this work were to investigate the effects of unconventional oils rich in phenolic compounds and rumen adaption on methane (CH4) gas production and rumen fermentation characteristics under in vitro rumen conditions. For this purpose, two sets of trials were conducted. In the first trial, the effects of blackberry, blueberry, raspberry, pomegranate, black seed and hemp oils on CH4 production and fermentation were examined in three 24 h batch culture experiments. Treatments in each experiment consisted of control (no oil supplement), control plus corn oil, or control plus two of the unconventional oils. Oils were added to rumen cultures at 500 mg/L (equivalent to 3.3 g oil/kg of diet dry matter (DM)). After 24 h of incubation, CH4 production was not different between the control and the corn oil treatments. Of the six unconventional oils tested, only hemp and blueberry oils reduced (P\u3c0.05) CH4 production by 9-16% relative to the control and corn oil treatments. No significant differences were observed between treatments in dry matter digestibility (DMD) or total volatile fatty acids (tVFA). Except for a reduction (P\u3c0.05) in acetate concentration with the raspberry oil, and an increase (P\u3c0.05) in valerate concentration with the pomegranate oil, all other treatments had similar VFA concentrations. In the second trial, the effects of adding oregano essential oil (OEO) to adapted and unadapted rumen cultures on CH4 production and rumen fermentation were evaluated under in vitro condition. Rumen cultures were obtained from continues culture fermenters fed a control diet or control diet plus OEO at 250 mg/day for 10 days. The addition of OEO decreased (P\u3c0.05) ii CH4 production only in adapted cultures. Total VFA and acetate concentrations were greater (P\u3c0.05) in the unadapted than adapted cultures and their concentrations decreased (P\u3c0.05) with the addition of OEO particularly when added to the adapted cultures. Propionate concentrations were also greater (P\u3c0.05) in the unadapted than the adapted cultures and concentrations decreased (P\u3c0.05) with the addition of OEO. Dry matter degradability and total gas production decreased (P\u3c0.03) with the addition of OEO in both cultures and total gas production tended (P\u3c0.13) to be lower when added to the adapted cultures. In conclusion, our results showed that hemp and blueberry oils were moderately effective in reducing rumen CH4 formation without compromising rumen fermentation and digestibility. Oregano Essential oil addition negatively affected rumen fermentation in both adapted and unadapted cultures and the effect was greater in the adapted cultures. The greater effects of OEO on CH4 production in the adapted cultures most likely due to the lower fermentation efficiency in these cultures

Characterization of Pgp3, a Chlamydia trachomatis Plasmid-Encoded Immunodominant Antigen▿

Human antibody recognition of Chlamydia trachomatis plasmid-encoded Pgp3 protein is dependent on the native conformation of Pgp3. The structural basis for the conformation dependence and the function of Pgp3 remain unknown. Here, we report that Pgp3 trimerization is required for the recognition of Pgp3 by human antibodies. In a native polyacrylamide gel, Pgp3 purified from a bacterial expression system migrated as stable trimers that were dissociated into monomers only by treatment with urea or sodium dodecyl sulfate (SDS) but not nonionic detergents. Human antibodies recognized trimeric but not monomeric Pgp3, suggesting that Pgp3 is presented to the human immune system as trimers during C. trachomatis infection. The endogenous Pgp3 secreted into the chlamydial outer membrane complex or host cell cytosol is always trimerized. Intact Pgp3 trimers were eluted from the outer membrane complex by a combination of nonionic detergents with reducing agents but not by the presence of either alone. These observations have provided important information for further understanding the role of Pgp3 in chlamydial pathogenesis and potentially optimizing Pgp3 as a subunit vaccine candidate antigen

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the strain of the conformer that is propagating

Unidee 2013 : the Third Paradise II

"The Third Paradise II is a compilation of reflections, thoughts and comments by UNIDEE 2013 residents, that aims to establish a dialogue with the institution and its conceptual and material constitution." -- Back cover

Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection.

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the "strain" of the conformer that is propagating