Biomedical subjects

Jan Bieschke

Publications and source records attributed to Jan Bieschke.

11 recordsLinked to original sources

PubMed2006

Amide-to-E-olefin versus amide-to-ester backbone H-bond perturbations: Evaluating the O-O repulsion for extracting H-bond energies.

Both amide-to-ester and amide-to-E-olefin backbone amide mutation methods were employed to perturb the same H-bond (formed by the NH of F23 and the CO of R14) in the Pin WW domain. Comparison of the thermodynamic folding energies of the ester mutant and the E-olefin mutant, accounting for the transfer free energy differences measured on relevant model compounds, yielded an estimated value of 0.3 kcal/mol for the O-O repulsion term (DeltaGO-Orep) in a beta-sheet context. The value of DeltaGO-Orep enabled us to calculate the intrinsic F23-R14 H-bond free energy to be 1.3 kcal/mol.

Alkenes↗

PubMed2006

Opposing activities protect against age-onset proteotoxicity.

Aberrant protein aggregation is a common feature of late-onset neurodegenerative diseases, including Alzheimer's disease, which is associated with the misassembly of the Abeta(1-42) peptide. Aggregation-mediated Abeta(1-42) toxicity was reduced in Caenorhabditis elegans when aging was slowed by decreased insulin/insulin growth factor-1-like signaling (IIS). The downstream transcription factors, heat shock factor 1, and DAF-16 regulate opposing disaggregation and aggregation activities to promote cellular survival in response to constitutive toxic protein aggregation. Because the IIS pathway is central to the regulation of longevity and youthfulness in worms, flies, and mammals, these results suggest a mechanistic link between the aging process and aggregation-mediated proteotoxicity.

Aging↗

PubMed2006

Structure-function-folding relationship in a WW domain.

Protein folding barriers result from a combination of factors including unavoidable energetic frustration from nonnative interactions, natural variation and selection of the amino acid sequence for function, and/or selection pressure against aggregation. The rate-limiting step for human Pin1 WW domain folding is the formation of the loop 1 substructure. The native conformation of this six-residue loop positions side chains that are important for mediating protein-protein interactions through the binding of Pro-rich sequences. Replacement of the wild-type loop 1 primary structure by shorter sequences with a high propensity to fold into a type-I' beta-turn conformation or the statistically preferred type-I G1 bulge conformation accelerates WW domain folding by almost an order of magnitude and increases thermodynamic stability. However, loop engineering to optimize folding energetics has a significant downside: it effectively eliminates WW domain function according to ligand-binding studies. The energetic contribution of loop 1 to ligand binding appears to have evolved at the expense of fast folding and additional protein stability. Thus, the two-state barrier exhibited by the wild-type human Pin1 WW domain principally results from functional requirements, rather than from physical constraints inherent to even the most efficient loop formation process.

Amino Acid Sequence↗

PubMed2006

Small molecule oxidation products trigger disease-associated protein misfolding.

Oxidative stress and inflammation are risk factors for both the development of alpha-synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, and Alzheimer's disease, the two most common neurodegenerative disorders. These diseases are associated with the neurotoxic deposition of misassembled alpha-synuclein and amyloid-beta (Abeta) peptides, respectively. Both occur sporadically, that is, without detectable disease-related mutations, in the vast majority of cases. Small molecule oxidation products, especially secosterols derived from cholesterol and 4-hydroxynonenal derived from lipid peroxidation, found in afflicted brains, accelerate the misassembly of both Abeta and alpha-synuclein. This Account explores the mechanism of small molecule oxidation product-mediated protein misassembly and possible intervention strategies.

Aldehydes↗

PubMed2005

E-olefin dipeptide isostere incorporation into a polypeptide backbone enables hydrogen bond perturbation: probing the requirements for Alzheimer's amyloidogenesis.

Herein, we report a stereospecific E-olefin dipeptide isostere synthesis that can be used to make gram quantities of the Phe-Phe isostere desired for eliminating a specific backbone H-bond donor and acceptor in the Alzheimer's disease related Abeta peptide. The Phe19-Phe20 E-olefin analogue of Abeta(1-40) was prepared by solid-phase peptide synthesis and was subjected to amyloidogenesis conditions. This analogue can aggregate into spherical morphologies but does not progress on to form protofibrils or fibrils as is the case for the all-amide sequence, providing insight into the structural requirements for amyloidogenesis.

Alkenes↗

PubMed2005

Single particle detection and characterization of synuclein co-aggregation.

Protein aggregation is the key event in a number of human diseases such as Alzheimer's and Parkinson's disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type alpha-synuclein forms co-aggregates with a mutant variant found in familial Parkinson's disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects.

Amyloid↗

PubMed2005

Automated PrPres amplification using indirect sonication.

Prions, which mainly consist of the scrapie isoform of the prion protein (PrP(Sc)), induce the misfolding of the physiological prion protein (PrP(C)). The Protein Misfolding Cyclic Amplification (PMCA), a process consisting of sonication and incubation, is one of the few methods thought to model autocatalytic prion replication and generation of proteinase K (PK)-resistant PrP (PrPres) in vitro. Here we show for the first time that the amplification may be achieved through direct as well as indirect sonication (water bath sonication using sealed sample containers), allowing the PMCA method to be automated. The automated method may serve as a valuable tool in high throughput screening for the diagnosis or compound identification for treatment of prion disease. The in vitro amplification process is weakly facilitated by divalent cations such as Mn, Zn and Ni, but not Cu, however, the presence of metal ions decreases the stability of PrPres against proteinase K digestion.

Animals↗

PubMed2005

Oxidative metabolites accelerate Alzheimer's amyloidogenesis by a two-step mechanism, eliminating the requirement for nucleation.

The process of amyloid formation by the amyloid beta peptide (Abeta), i.e., the misassembly of Abetapeptides into soluble quaternary structures and, ultimately, amyloid fibrils, appears to be at the center of Alzheimer's disease (AD) pathology. We have shown that abnormal oxidative metabolites, including cholesterol-derived aldehydes, modify Abeta and accelerate the early stages of amyloidogenesis (the formation of spherical aggregates). This process, which we have termed metabolite-initiated protein misfolding, could explain why hypercholesterolemia and inflammation are risk factors for sporadic AD. Herein, the mechanism by which cholesterol metabolites hasten Abeta 1-40 amyloidogenesis is explored, revealing a process that has at least two steps. In the first step, metabolites modify Abeta peptides by Schiff base formation. The Abeta-metabolite adducts form spherical aggregates by a downhill polymerization that does not require a nucleation step, dramatically accelerating Abeta aggregation. In agitated samples, a second step occurs in which fibrillar aggregates form, a step also accelerated by cholesterol metabolites. However, the metabolites do not affect the rate of fibril growth in seeded aggregation assays; their role appears to be in initiating amyloidogenesis by lowering the critical concentration for aggregation into the nanomolar range. Small molecules that block Schiff base formation inhibit the metabolite effect, demonstrating the importance of the covalent adduct. Metabolite-initiated amyloidogenesis offers an explanation for how Abeta aggregation could occur at physiological nanomolar concentrations.

Alzheimer Disease↗

PubMed2005

Systematic identification of antiprion drugs by high-throughput screening based on scanning for intensely fluorescent targets.

Conformational changes and aggregation of specific proteins are hallmarks of a number of diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. In the case of prion diseases, the prion protein (PrP), a neuronal glycoprotein, undergoes a conformational change from the normal, mainly alpha-helical conformation to a disease-associated, mainly beta-sheeted scrapie isoform (PrP(Sc)), which forms amyloid aggregates. This conversion, which is crucial for disease progression, depends on direct PrP(C)/PrP(Sc) interaction. We developed a high-throughput assay based on scanning for intensely fluorescent targets (SIFT) for the identification of drugs which interfere with this interaction at the molecular level. Screening of a library of 10,000 drug-like compounds yielded 256 primary hits, 80 of which were confirmed by dose response curves with half-maximal inhibitory effects ranging from 0.3 to 60 microM. Among these, six compounds displayed an inhibitory effect on PrP(Sc) propagation in scrapie-infected N2a cells. Four of these candidate drugs share an N'-benzylidene-benzohydrazide core structure. Thus, the combination of high-throughput in vitro assay with the established cell culture system provides a rapid and efficient method to identify new antiprion drugs, which corroborates that interaction of PrP(C) and PrP(Sc) is a crucial molecular step in the propagation of prions. Moreover, SIFT-based screening may facilitate the search for drugs against other diseases linked to protein aggregation.

Animals↗

PubMed2004

Autocatalytic self-propagation of misfolded prion protein.

Prions are thought to replicate in an autocatalytic process that converts cellular prion protein (PrP(C)) to the disease-associated misfolded PrP isoform (PrP(Sc)). Our study scrutinizes this hypothesis by in vitro protein misfolding cyclic amplification (PMCA). In serial transmission PMCA experiments, PrP(Sc) was inoculated into healthy hamster brain homogenate containing PrP(C). Misfolded PrP was amplified by rounds of sonication and incubation and reinoculated into fresh brain homogenate every 10 PMCA rounds. The amplification depended on PrP(C) substrate and could be inhibited by recombinant hamster PrP. In serial dilution experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalyzed the structural conversion of PrP(C) as efficiently as PrP(Sc) from brain of scrapie (263K)-infected hamsters, yielding an approximately 300-fold total amplification of PrPres after 100 rounds, which confirms an autocatalytic PrP-misfolding cascade as postulated by the prion hypothesis. PrPres formation was not paralleled by replication of biological infectivity, which appears to require factors additional to PrP-misfolding autocatalysis.

Animals↗

PubMed2004

Metabolite-initiated protein misfolding may trigger Alzheimer's disease.

Anfinsen showed that a protein's fold is specified by its sequence. Although it is clear why mutant proteins form amyloid, it is harder to rationalize why a wild-type protein adopts a native conformation in most individuals, but it misfolds in a minority of others, in what should be a common extracellular environment. This discrepancy suggests that another event likely triggers misfolding in sporadic amyloid disease. One possibility is that an abnormal metabolite, generated only in some individuals, covalently modifies the protein or peptide and causes it to misfold, but evidence for this is sparse. Candidate metabolites are suggested by the recently appreciated links between Alzheimer's disease (AD) and atherosclerosis, known chronic inflammatory metabolites, and the newly discovered generation of ozone during inflammation. Here we report detection of cholesterol ozonolysis products in human brains. These products and a related, lipid-derived aldehyde covalently modify Abeta, dramatically accelerating its amyloidogenesis in vitro, providing a possible chemical link between hypercholesterolemia, inflammation, atherosclerosis, and sporadic AD.

Alzheimer Disease↗