Determination of protein complex stoichiometry through multisignal sedimentation velocity experiments

Determination of the stoichiometry of macromolecular assemblies is fundamental to an understanding of how they function. Many different biophysical methodologies may be used to determine stoichiometry. In the past, both sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation have been employed to determine component stoichiometries. Recently, a method of globally analyzing multisignal sedimentation velocity data was introduced by Schuck and coworkers. This global analysis removes some of the experimental inconveniences and inaccuracies that could occur in the previously used strategies. This method uses spectral differences between the macromolecular components to decompose the well-known c(s) distribution into component distributions c_k(s); that is, each component k has its own c_k(s) distribution. Integration of these distributions allows the calculation of the populations of each component in cosedimenting complexes, yielding their stoichiometry. In our laboratories, we have used this method extensively to determine the component stoichiometries of several protein-protein complexes involved in cytoskeletal remodeling, sugar metabolism, and host-pathogen interactions. The overall method is described in detail in this work, as are experimental examples and caveats.     

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden

Background

It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.

Results

Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember?, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced.

Conclusions

Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

     

Direct and indirect food web regulation of microbial decomposers in headwater streams

The direct and indirect regulation of primary productivity has been well established in autotrophic‐based ecosystems; however, less is known about the processes affecting decomposers in detrital‐based ecosystems. Because, small headwater, woodland streams are a dominate feature in most ecosystems and are tightly linked to terrestrial detritus, understanding decomposer‐mediated functions in these systems is critical for understanding carbon processes across the landscape. In this light, we conducted a microcosm and mesocosm experiment to test the direct and indirect food web effects on decomposers in small stream ecosystems. The results from the microcosm experiment supported an existing literature, demonstrating that nutrients directly stimulate decomposers and that microbivores directly reduce decomposers. Based on well‐founded food web theory in autotrophic systems, we predicted that fishes from different trophic‐functional guilds would indirectly stimulate decomposers by enhancing dissolved nutrients and by reducing microbivore densities. Our mesocosm experiment partially supported these predictions. Specifically, we found that fishes that consumed mostly terrestrial foods increased decomposers from the bottom–up by enhancing allochthonous nutrient loading into the stream ecosystems. Contrary to our predictions, however, predatory fishes that consume microbivores did not increase decomposers from the top–down. Rather, in streams with the predatory fish species, microbivores increased (rather than decreased) on leaf litter. This may have resulted from an experimental artifact associated with refuge provided by leaf packs. In conclusion, our data demonstrate that decomposers are regulated by similar direct and indirect processes important in autotrophic‐based ecosystems. This provides further evidence that food web processes can regulate leaf decomposition and flux of detrital carbon through ecosystems.     

Low utilization of circulating glucose after food withdrawal in Snell dwarf mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.     

RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity

Carpenter syndrome is a pleiotropic disorder with autosomal recessive inheritance, the cardinal features of which include craniosynostosis, polysyndactyly, obesity, and cardiac defects. Using homozygosity mapping, we found linkage to chromosome 6p12.1-q12 and, in 15 independent families, identified five different mutations (four truncating and one missense) in RAB23, which encodes a member of the RAB guanosine triphosphatase (GTPase) family of vesicle transport proteins and acts as a negative regulator of hedgehog (HH) signaling. In 10 patients, the disease was caused by homozygosity for the same nonsense mutation, L145X, that resides on a common haplotype, indicative of a founder effect in patients of northern European descent. Surprisingly, nonsense mutations of Rab23 in open brain mice cause recessive embryonic lethality with neural-tube defects, suggesting a species difference in the requirement for RAB23 during early development. The discovery of RAB23 mutations in patients with Carpenter syndrome implicates HH signaling in cranial-suture biogenesis--an unexpected finding, given that craniosynostosis is not usually associated with mutations of other HH-pathway components--and provides a new molecular target for studies of obesity.     

Genomewide scan for linkage reveals evidence of several susceptibility loci for alopecia areata

Alopecia areata (AA) is a genetically determined, immune-mediated disorder of the hair follicle that affects 1%-2% of the U.S. population. It is defined by a spectrum of severity that ranges from patchy localized hair loss on the scalp to the complete absence of hair everywhere on the body. In an effort to define the genetic basis of AA, we performed a genomewide search for linkage in 20 families with AA consisting of 102 affected and 118 unaffected individuals from the United States and Israel. Our analysis revealed evidence of at least four susceptibility loci on chromosomes 6, 10, 16 and 18, by use of several different statistical approaches. Fine-mapping analysis with additional families yielded a maximum multipoint LOD score of 3.93 on chromosome 18, a two-point affected sib pair (ASP) LOD score of 3.11 on chromosome 16, several ASP LOD scores >2.00 on chromosome 6q, and a haplotype-based relative risk LOD of 2.00 on chromosome 6p (in the major histocompatibility complex locus). Our findings confirm previous studies of association of the human leukocyte antigen locus with human AA, as well as the C3H-HeJ mouse model for AA. Interestingly, the major loci on chromosomes 16 and 18 coincide with loci for psoriasis reported elsewhere. These results suggest that these regions may harbor gene(s) involved in a number of different skin and hair disorders.     

Rational design of 6-(2,4-diaminopyrimidinyl)-1,4-benzoxazin-3-ones as small molecule renin inhibitors

We report the design and synthesis of a series of 6-(2,4-diaminopyrimidinyl)-1,4-benzoxazin-3-ones as orally bioavailable small molecule inhibitors of renin. Compounds with a 2-methyl-2-aryl substitution pattern exhibit potent renin inhibition and good permeability, solubility, and metabolic stability. Oral bioavailability was found to be dependent on metabolic clearance and cellular permeability, and was optimized through modulation of the sidechain that binds in the S3(sp) subsite.