9 research outputs found
Proteolytically inactive insulin-degrading enzyme inhibits amyloid formation yielding non-neurotoxic aβ peptide aggregates.
Insulin-degrading enzyme (IDE) is a neutral Zn(2+) peptidase that degrades short peptides based on substrate conformation, size and charge. Some of these substrates, including amyloid β (Aβ) are capable of self-assembling into cytotoxic oligomers. Based on IDE recognition mechanism and our previous report of the formation of a stable complex between IDE and intact Aβ in vitro and in vivo, we analyzed the possibility of a chaperone-like function of IDE. A proteolytically inactive recombinant IDE with Glu111 replaced by Gln (IDEQ) was used. IDEQ blocked the amyloidogenic pathway of Aβ yielding non-fibrillar structures as assessed by electron microscopy. Measurements of the kinetics of Aβ aggregation by light scattering showed that 1) IDEQ effect was promoted by ATP independent of its hydrolysis, 2) end products of Aβ-IDEQ co-incubation were incapable of "seeding" the assembly of monomeric Aβ and 3) IDEQ was ineffective in reversing Aβ aggregation. Moreover, Aβ aggregates formed in the presence of IDEQ were non-neurotoxic. IDEQ had no conformational effects upon insulin (a non-amyloidogenic protein under physiological conditions) and did not disturb insulin receptor activation in cultured cells. Our results suggest that IDE has a chaperone-like activity upon amyloid-forming peptides. It remains to be explored whether other highly conserved metallopeptidases have a dual protease-chaperone function to prevent the formation of toxic peptide oligomers from bacteria to mammals
Effect of IDEQ upon solubility and secondary structure of Aβ aggregates.
<p>(<b>A</b>) samples containing Aβ1-42 before incubation (“dead time”) and after incubation for 5 days with or without IDEQ were centrifuged at 3,000× g for 5 min and supernatants and pellets analyzed by Western blots with anti-Aβ 6E10 and 4G8. Arrowheads indicate: H, high molecular mass oligomers, T, Aβ tetramers and <i>t</i>, Aβ trimers. (<b>B</b>) Densitometric quantification of Aβ1-42 obtained from Western blots shown in panel (<b>A</b>). Bars represent the mean ± SEM of total Aβ immunoreactivity in arbitrary units (AU). * p<0.05, Student's <i>t</i> test. (<b>C</b>) Far UV-CD spectra recorded at “dead time” of Aβ1-42 (solid black line), IDEQ alone (dotted line) and Aβ1-42 co-incubated with IDEQ (solid gray line) at a 1∶300 molar ratio (IDEQ:Aβ). (<b>D</b>) Far UV-spectra recorded after 6 days of incubation of Aβ1-42 alone or Aβ1-42 with IDEQ at 1∶300 molar ratio. Samples were centrifuged as described above and supernatants analyzed. IDEQ alone, dotted line; Aβ1-42 alone, solid black line; Aβ1-42 co-incubated with IDEQ, solid gray line.</p
Aβ1-42 oligomers formed in the presence of IDEQ are not neurotoxic.
<p>(<b>A</b>) Representative AFM images showing the size and morphology of Aβ1-42 neurotoxic species. Left, Aβ1-42 incubated alone for 4 days. Approximately spherical species of ∼20–30 nm are indicated by arrowheads. Rods and short protofibrils are depicted by arrows. Inset: a larger Aβ1-42 protofibril is shown. Right; Aβ1-42 incubated in the presence of IDEQ at a 1∶10 molar ratio (IDEQ: Aβ) showing larger aggregates of 50–60 nm (arrowheads) and rods with lengths of ∼100–120 nm (arrows). (<b>B</b>) Representative immunofluorescence of primary differentiated neurons exposed to vehicle, Aβ1-42 alone or Aβ1-42 pre-incubated with IDEQ from top to bottom, as indicated. White arrows point at neuronal processes. Bars = 30 μM. (<b>C</b>) Analysis of neuronal processes under the conditions as shown in panel (<b>A</b>). Bars represent the mean ± SEM of processes' lengths as measured from the centre of the neuronal body * p<0.01, one-way ANOVA, Tukey post-hoc test. (<b>D</b>) Viability of mature primary neurons after the indicated treatments as assessed by MTT reduction. * p<0.05, one-way ANOVA, Tukey post-hoc test. Results are shown for three independent experiments.</p
Effect of IDEQ and ATP upon the kinetic of Aβ aggregation and seeding.
<p>(<b>A</b>) Turbidity profiles of Aβ1-42 at 15 μM alone in working buffer (▴) or in the presence of IDEQ at the indicated molar ratios (IDEQ:Aβ), from top to bottom: 1∶200 (□), 1∶100 (Δ), 1∶10 (◯) and 1∶10 containing 0.5 mM ATP (<b>•</b>). Light scattering at 340 nm was measured every 30 min using a TECAN GENios multi-well reader (for clarity, only the points every other 90 min are shown). The bracket encloses the curves obtained after co-incubation of Aβ1-42 with IDEQ at the indicated conditions. Results are expressed as mean ± S.E.M. of at least two independent experiments in duplicate. (<b>B</b>) Representative TEM images of samples at steady state of Aβ1-42 alone (top) or with IDEQ at 1∶10 molar ratio in the presence of ATP (bottom). Bars = 100 nm. (<b>C</b>) Time course of Aβ1-42 aggregation alone (▴), in the presence of seeds previously formed with IDEQ (◯), or after the addition of pure Aβ1-42 seeds (□). (<b>D</b>) Kinetics of aggregation of Aβ1-42 after the addition of IDEQ (◯) or the same volume of working buffer (▴) to Aβ1-42 after 48 h of self-assembly, as indicated by the arrow.</p
IDEQ does not modify insulin conformation.
<p>(<b>A</b>) <i>Circular dichroism (CD) spectra</i> of 10 μM insulin (ins.) in working buffer (solid black line), insulin with IDEQ at 1∶100 molar ratio, enzyme:insulin (solid gray line) and IDEQ alone (dotted line) with no prior incubation. (<b>B</b>) Same samples as in panel (<b>A</b>) after incubation for 24 h at 25°C. Insulin alone (solid black line), insulin with IDEQ (solid gray line) and IDEQ alone (dotted line). (<b>C</b>) Western blot with anti-phospho-Akt and anti-total Akt of U-87 cell lysates. Cells were exposed for 30 min with insulin alone, insulin previously co-incubated with IDEwt or IDEQ, as indicated. Wortmannin (wort) was incubated at 10 nM for 30 min before treatments.</p
Time-course, amount and pH-dependence of the formation of complexes between Aβ1-42 and IDEwt or IDEQ.
<p>(<b>A</b>) Western blots with anti-Aβ 6E10 showing the ∼120 kDa band corresponding to IDE-AβSCx (IDE-Aβ stable complex) as a function of the incubation time. Top panel, IDEQ; lower panel, IDEwt. Both PVDF membranes were developed simultaneously with a STORM 860 scanner. Below each Western blot, the same membranes stained with Coomassie blue, show IDEwt or IDEQ loading. (<b>B</b>) Densitometric data from Western blots for IDEQ (◯) and IDEwt (▴) were fitted to a single exponential equation using Graph Pad Prism v.4 software. Points represent the mean ± SEM from two independent experiments in duplicate. (<b>C</b>) IDEQ-AβSCx formation is partially competed by pre-incubation for 1 h with insulin at the indicated molar excess before the addition of Aβ1-42. Data are expressed as the percentage of the remaining Aβ-positive band at ∼120 kDa, in arbitrary units, as a function of insulin concentration. Each point represents the mean ± SEM of two independent experiments in duplicate. Inset: a representative Western blot of IDEQ-AβSCx developed with 6E10. (<b>D</b>) Densitometry of IDEQ-AβSCx at the indicated range of pH as determined by Western blot with anti-Aβ. Bars represent the mean ± SEM of three separate experiments. Inset: top, representative Western blot with anti-Aβ of IDEQ-AβSCx; bottom, Coomassie blue of IDEQ loaded in each lane. In panels (<b>A</b>), (<b>C</b>) and (<b>D</b>), IDEwt or IDEQ-AβSCxs are indicated by arrowheads.</p
Proteolytically Inactive Insulin-Degrading Enzyme Inhibits Amyloid Formation Yielding Non-Neurotoxic Aβ Peptide Aggregates
The Role of Finance in Economic Development: Benefits, Risks, and Politics
Abstract: Theoretical and empirical research has shown that a sound and effective financial system is critical for economic development and growth. The financial system, however, is also subject to boom and bust cycles and fragility, with negative repercussions for the real economy. Further, the political structure of societies, often pre-determined by historic experience, is critical for the structure and development of the financial system. This paper is a critical survey of three related strands of literature – the finance and growth literature, the literature on financial fragility, and the politics and finance literature.
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field