Research Report 2010 - MDC

Structure of the GroupJan Bieschke(Delbrück Fellow)Group LeaderDr. Jan BieschkeScientistsDr. Ralf FriedrichGraduate StudentsKatja WelschMaliha Shah*Heike Wobst*(Master student)Technical AssistantsGerlinde GrelleSusanne Kostka*part of the periodreportedAging-related Protein Misfolding andDetoxification MechanismsThe misfolding of endogenous protein or peptide fragments to form cytotoxic depositsmade from fibrillar protein aggregates characterizes amyloid diseases. The misfoldedpolypeptides, which are believed to be the root cause of the pathologies, are specific for eachdisease, such as amyloid β (Aβ) in Alzheimer’s disease (AD), α-synuclein in Parkinson’s disease(PD) or fragments of the huntingtin protein in Huntigton’s disease (HD). The specific natureand mechanism of the cytotoxicity are yet unknown. However, a wealth of evidence points tosmaller oligomeric aggregates rather than large fibrils bein the crucial species.Both AD and PD mostly occur sporadically without knowngenetic components. They have the patient’s age as thesingle most important risk factor. Yet, little is known howaging influences them. Our lead hypothesis states thattoxic protein aggregates result from an imbalance in thedynamic equilibrium between aggregation and clearanceof misfolded proteins rather than from a slow stochasticprocess, as has previously been assumed.The mechanistic details of protein misfolding, the autocatalyticreplication of misfolded protein aggregates,and possible detoxifying mechanisms are the majorpoints of our research (Fig. A). We found that misfoldedAβ aggregates are detoxified by two opposing activitiesunder the control of two interconnected aging-relatedpathways, the Insulin/IGF-receptor pathway on onehand and the stress response / heat shock reponsepathways controlled by HSF-1 on the other hand (Fig B).The primary detoxification pathway appears to involvedisassembly of misfolded aggregates, a secondary pathwaythe induced aggregation of small Aβ aggregatesinto larger, less-toxic structures.Endogenous Modifiers of Amyloid FormationWe aim to identify key components of the cellulardetoxification machinery and study the influence ofendogenous proteins on aggregate assembly and disassemblyusing several screening based methods:a. Cellular aggregation assays and siRNAinterferenceKatja WelschAggregation-prone fragments of huntingtin areexpressed in mammalian cells (N2A, PC12). Target genesfrom a library of proteins that are related to proteinfolding or to the aging pathways are simultaneouslydownregulated by siRNA during aggregation or afterthe completion of aggregation to assess their influenceon amyloid formation and removal by the cells.b. Genome-wide functional screening foramyloid modulatorsRalf Friedrich, Maliha ShahProteins are recombinantly expressed from a genomichuman protein library (ca. 14.000 constructs) and addedto in vitro aggregation assays and disaggregationassays (Fig C, D) using the Aβ peptides and theα-synuclein protein. Aggregation and disaggregationkinetics are used to identify proteins that influenceaggregation or that have a disaggregating activity and186 Function and Dysfunction of the Nervous System