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- Caulfield, Thomas R., et al.
(författare)
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Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin
- 2014
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Ingår i: PloS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358. ; 10:11, s. e1003935-
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Tidskriftsartikel (refereegranskat)abstract
- Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimalbiasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.
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| 3. |
- Fiesel, Fabienne C., et al.
(författare)
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Structural and Functional Impact of Parkinson Disease-Associated Mutations in the E3 Ubiquitin Ligase Parkin
- 2015
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Ingår i: Human Mutation. - : Hindawi Limited. - 1059-7794 .- 1098-1004. ; 36:8, s. 774-786
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the PARKIN/PARK2 gene that result in loss-of-function of the encoded, neuroprotective E3 ubiquitin ligase Parkin cause recessive, familial early-onset Parkinson disease. As an increasing number of rare Parkin sequence variants with unclear pathogenicity are identified, structure-function analyses will be critical to determine their disease relevance. Depending on the specific amino acids affected, several distinct pathomechanisms can result in loss of Parkin function. These include disruption of overall Parkin folding, decreased solubility, and protein aggregation. However pathogenic effects can also result from misregulation of Parkin autoinhibition and of its enzymatic functions. In addition, interference of binding to coenzymes, substrates, and adaptor proteins can affect its catalytic activity too. Herein, we have performed a comprehensive structural and functional analysis of 21 PARK2 missense mutations distributed across the individual protein domains. Using this combined approach, we were able to pinpoint some of the pathogenic mechanisms of individual sequence variants. Similar analyses will be critical in gaining a complete understanding of the complex regulations and enzymatic functions of Parkin. These studies will not only highlight the important residues, but will also help to develop novel therapeutics aimed at activating and preserving an active, neuroprotective form of Parkin.
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- Sandström, Niklas, 1981-, et al.
(författare)
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Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip
- 2022
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Ingår i: CELL REPORTS METHODS. - : Elsevier BV. - 2667-2375. ; 2:7
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Tidskriftsartikel (refereegranskat)abstract
- Here, we present a methodology based on multiplexed fluorescence screening of two-or three-dimensional cell cultures in a newly designed multichambered microwell chip, allowing direct assessment of drug or im-mune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluores-cence labeling, and imaging of fixed or live cells at various magnifications directly in the chip together with data analysis and interpretation. The methodology is demonstrated by drug cytotoxicity screening using ovarian and non-small cell lung cancer cells and by cellular cytotoxicity screening targeting tumor spheroids of renal carcinoma and ovarian carcinoma with natural killer cells from healthy donors. The miniaturized format allowing long-term cell culture, efficient screening, and high-quality imaging of small sample volumes makes this methodology promising for individualized cytotoxicity tests for precision medicine.
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