The Effect of Ferrocene Derivatives on the Thermal Degradation of Polyethylene

The inhibiting action of ferrocene and p-ferrocenyl-aniline on the kinetics of thermal degradation of polyethylene was investigated by gravimetry, by measuring the changes in average molecular weight, by gas chromatography and spectroscopic techniques. The effect of ferrocene .derivatives is explained by assuming chemical reactions of the decomposition products (such as cyclopentadiene and cyclopentadienyl radicals) with macroradicals to occur

Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation

Evaluation of SMN Protein, Transcript, and Copy Number in the Biomarkers for Spinal Muscular Atrophy (BforSMA) Clinical Study

BACKGROUND: The universal presence of a gene (SMN2) nearly identical to the mutated SMN1 gene responsible for Spinal Muscular Atrophy (SMA) has proved an enticing incentive to therapeutics development. Early disappointments from putative SMN-enhancing agent clinical trials have increased interest in improving the assessment of SMN expression in blood as an early "biomarker" of treatment effect. METHODS: A cross-sectional, single visit, multi-center design assessed SMN transcript and protein in 108 SMA and 22 age and gender-matched healthy control subjects, while motor function was assessed by the Modified Hammersmith Functional Motor Scale (MHFMS). Enrollment selectively targeted a broad range of SMA subjects that would permit maximum power to distinguish the relative influence of SMN2 copy number, SMA type, present motor function, and age. RESULTS: SMN2 copy number and levels of full-length SMN2 transcripts correlated with SMA type, and like SMN protein levels, were lower in SMA subjects compared to controls. No measure of SMN expression correlated strongly with MHFMS. A key finding is that SMN2 copy number, levels of transcript and protein showed no correlation with each other. CONCLUSION: This is a prospective study that uses the most advanced techniques of SMN transcript and protein measurement in a large selectively-recruited cohort of individuals with SMA. There is a relationship between measures of SMN expression in blood and SMA type, but not a strong correlation to motor function as measured by the MHFMS. Low SMN transcript and protein levels in the SMA subjects relative to controls suggest that these measures of SMN in accessible tissues may be amenable to an "early look" for target engagement in clinical trials of putative SMN-enhancing agents. Full length SMN transcript abundance may provide insight into the molecular mechanism of phenotypic variation as a function of SMN2 copy number. TRIAL REGISTRY: Clinicaltrials.gov NCT00756821

Amyloid-Mediated Sequestration of Essential Proteins Contributes to Mutant Huntingtin Toxicity in Yeast

BACKGROUND: Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN+]), which has a glutamine/asparagine-rich domain. PRINCIPAL FINDINGS: Here, we showed that aggregation and toxicity of mutant htt depended on [PIN+] only quantitatively: the presence of [PIN+] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN+], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN+] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast. CONCLUSIONS: The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity

Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI+]

Prions are proteins that can access multiple conformations, at least one of which is beta-sheet rich, infectious and self-perpetuating in nature. These infectious proteins show several remarkable biological activities, including the ability to form multiple infectious prion conformations, also known as strains or variants, encoding unique biological phenotypes, and to establish and overcome prion species (transmission) barriers. In this Perspective, we highlight recent studies of the yeast prion [PSI+], using various biochemical and structural methods, that have begun to illuminate the molecular mechanisms by which self-perpetuating prions encipher such biological activities. We also discuss several aspects of prion conformational change and structure that remain either unknown or controversial, and we propose approaches to accelerate the understanding of these enigmatic, infectious conformers

Modeling Spinal Muscular Atrophy in Drosophila

Spinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. We used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Here we show that a normal neuromuscular junction (NMJ) structure depends on SMN expression and that SMN concentrates in the post-synaptic NMJ regions. We conducted a screen for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that our genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets

Prion Formation and Polyglutamine Aggregation Are Controlled by Two Classes of Genes

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN+]. When fused to GFP and overexpressed in [ps−] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI+] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI+]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI+] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI+] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.National Institutes of Health (U.S.) (grant GM56350)National Institutes of Health (U.S.) (NSRA F32 postdoctoral fellowship GM072340)National Institutes of Health (U.S.) (grant GM25874)Howard Hughes Medical Institut

Molecular Determinants of Survival Motor Neuron (SMN) Protein Cleavage by the Calcium-Activated Protease, Calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN

[SWI+], the Prion Formed by the Chromatin Remodeling Factor Swi1, Is Highly Sensitive to Alterations in Hsp70 Chaperone System Activity

The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments