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Defining the pathways and mechanism of misfolded protein triage and quality control in eukaryotic cells

Final Report Summary - FOLDEG (Defining the pathways and mechanism of misfolded protein triage and quality control in eukaryotic cells)

Maintaining a healthy proteome is essential for cellular fitness. Protein homeostasis (proteostasis) is regulated by protein quality control machineries, comprising molecular chaperons and protein clearance systems. The molecular chaperones recognize misfolded proteins (partially induced by gene mutations), aim to help them obtaining their native conformation required for proper function or target them for cellular clearance. As a consequence of impaired proteostasis, misfolded proteins may accumulate in the cell and form cellular-toxic spatial misfolded, aggregation-prone protein deposition sites (e.g. JUNQ: juxta-nuclear quality control compartment; and IPOD: insoluble protein deposit). The activity and accuracy of the cellular protein quality control machineries are affected by aging, which may contribute to the aetiology and progression of aggregation-disease of the brain characterized by neurodegradation (including Huntington’s disease (HD)). It remains currently largely elusive how the quality control machineries recognize misfolded proteins, and how the triage decisions are implemented; i.e. why certain misfolded proteins are refolded whereas others are targeted for degradation or sequestration into cellular inclusions. Additionally, an intriguing question remaining is why neurons are particularly vulnerable to protein misfolding and (toxic) aggregation. This research project was aimed at identification of the principles and factors involved in the triage between protein folding and degradation in order to maintain a healthy proteome, and assessing the role of (co-)chaperones in quality control dysfunction linked to aging and aging-related neuropathologies such as HD.

The project has elucidated mechanisms underlying healthy brain aging and onset of protein misfolding and aggregation disorders, including HD, using original approaches that stand out from previous studies. The project has gained from insights from different disciplines, including neurobiology, stem cell biology, biophysics, and mouse genetics; as evident from the established national and international collaborations.
In particular, the project has identified a unique interplay and critical roles for HSP70 and other molecular chaperones in maintaining a healthy proteome under physiological conditions, a quality control pathway we named the Q-body pathway. Additionally, the project results demonstrate that the quality control networks are differentially regulated in distinct cell types, which may (partially) explain the high vulnerability of neuronal cells (in contrast to other cell types in the central nervous system) to protein misfolding and aggregation seen in late-onset neuropathologies (unpublished data).

Protein quality control triage
Protein misfolding, aggregation, and clearance was studied using various reporters of protein misfolding (reversible vs. irreversible misfolding; soluble vs. insoluble aggregate formation, etc.). The study demonstrated that in healthy, unstressed, cells, protein misfolding may occur resulting in sequestration into aggregates, demonstrated to be a highly dynamic process. Soluble, misfolded proteins recognized and processed by the identified quality control pathway are concentrated in dynamic structures (Q-bodies) that continue to merge into larger inclusions throughout the cell on route to degradation, primarily mediated by the ubiquitin-proteasome system. Q-body dynamics, coalescence, and clearance require ATP-dependent HSP70 and HSP90 chaperone function (with HSP70 being the main player). The activity of HSP70 and HSP90 is regulated by distinct the ER-anchored co-chaperones HSP40/J-domain proteins, Ydj1 and Hlj1. Additional HSPs (HSP104, and the small HSPs HSP26 and HSP42) play a major role in the Q-body pathway. Whereas HSP42 is required for Q-body formation, HSP104 promotes Q-body dissolution and maturation into larger inclusions by active coalescence. We were also able to demonstrate that the Q-body pathway does not overlap with the cellular processing of insoluble amyloidogenic IPOD substrates.

Protein aggregation in HD pathology
The protein aggregation process and quality control machineries were studied in detail in HD pathology. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several μm) globular neuronal inclusion bodies (IBs) over time. Aggregation of pathogenic Htt was studied in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. We demonstrated that Htt fibrils formed abundantly across the cytoplasmic compartment and also in neurite processes, yet only after nucleation and formation of a relatively large inclusion body (IB). Structural characterizations of fibrils by STED showed a distinct length cut off at ~1.5 μm and revealed subsequent coalescence (bundling/piling). Cytosolic fibrils were observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB. This has been argued to be a cell-protective phenomenon in neurons, presumably saturating and over-powering the cellular degradation systems and leaving cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further demonstrated that exogenous cellular delivery of the apical domain of the CCT1 subunit of the TRiC/CCT chaperonin complex (a chaperone capable to inhibit Htt aggregation in vitro and in vivo studies; consisting of CCT1-8) reduced the aggregation propensity of mutant Htt in general, though particularly strongly reduced the occurrence of the late-stage fibrils.


The fellow has been given an opportunity to learn from two successful female leading scientists in the field of protein quality control in health and disease. She has attained expertise and essential training in the field of proteostasis, expanded her teaching and mentoring skills by (co-)supervising BSc, MSc, and PhD students and research assistants, and gained professional maturity in grant writing as well as presenting her work at scientific meetings. She also successfully published her research results, initiated national and international collaborations, and received training financial and scientific research management.


There is a considerable interest in understanding how cellular proteostasis is achieved and contributes to preventing protein aggregation and neurotoxicity. With the increased incidence of late-onset protein aggregation diseases of the brain due to our increasing elderly population, and no availability of effective treatments to halt or cure theses late-onset pathologies, the results obtained in this study may not only provide insights into the various disease aetiologies, but also new avenues for therapeutic design to ultimately rejuvenate the aged and/or diseased brain. Also, as aging is a common determinant in many diseases characterized by aggregation or impaired proteostasis capacity (not limited to neurodegenerative diseases, but also include e.g. diabetes and cancer), the findings and concepts obtained from this project may also have implications for extending these fields of research. Overall, the obtained data (published, in submission, and in preparation) is extremely relevant to the fields studying proteostasis and neurobiology in the context of organismal aging.