I18-Microfocus Spectroscopy
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Rémi
Kinet
,
Joanna
Sikora
,
Marie-Laure
Arotcarena
,
Melina
Decourt
,
Eric
Balado
,
Evelyne
Doudnikoff
,
Sylvain
Bohic
,
Marta
Vesnaver
,
Anna
Lovisotto
,
Marie-Laure
Thiolat
,
Nathalie
Dutheil
,
Claire
Mazzocco
,
Karim
Harhouri
,
Rémy
Steinschneider
,
Severine
Menoret
,
Laurent
Tesson
,
Ignacio
Anegon
,
Michele
Morari
,
Miquel
Vila
,
François
Georges
,
Erwan
Bezard
,
Pierre-Olivier
Fernagut
,
Benjamin
Dehay
Diamond Proposal Number(s):
[29838]
Open Access
Abstract: Mutations in the ATP13A2 gene were identified as the cause of Kufor-Rakeb syndrome (KRS), a juvenile-onset form of Parkinson’s disease (PD). Developing relevant and predictive models for the rare PD forms is necessary to understand the pathological mechanisms and validate therapeutic strategies. Herein, we aimed to comprehensively characterize the first transgenic Atp13a2 knockout rat model. Behavioral assessment demonstrated specific developmental deficits in animals with deletion of Atp13a2. Further analysis revealed that Atp13a2 knockout rats displayed age-dependent fine motor skills deficits and impaired locomotor habituation similar to those observed in PD patients at the early stage of motor symptoms. In contrast, no change in the nigrostriatal integrity was observed. An extended investigation on heavy metals homeostasis, autophagy-related markers, and lipofuscin accumulation showed significant changes reminiscent of KRS. Finally, we tested whether inducing pathology by viral-mediated overexpression of human α-synuclein or human tyrosinase exacerbated the onset or extent of pathological changes. This Atp13a2 KO rat model could help better understand autophagy in PD pathogenesis and open new therapeutic validation opportunities.
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Nov 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[39148]
Open Access
Abstract: Frataxin is a 23 kDa mitochondrial iron-binding protein involved in the biogenesis of iron–sulfur (Fe–S) clusters. Deficiency in frataxin is associated with Friedreich's ataxia, a progressive neurodegenerative disorder. CyaY, the bacterial ortholog of eukaryotic frataxin, is believed to function as an iron donor in Fe–S cluster assembly, making it a key target for structural and functional studies. In this work, a comprehensive structural analysis of the Escherichia coli CyaY protein is presented, comparing its structure at room temperature and cryogenic conditions. Notably, the first room-temperature structures are obtained using the Turkish Light Source “Turkish DeLight” X-ray diffractometer and serial synchrotron X-ray crystallography, marking a significant step forward in understanding CyaY under near-physiological conditions.
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Oct 2025
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I14-Hard X-ray Nanoprobe
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Gaewyn
Ellison
,
Rhiannon E.
Boseley
,
Meg
Willans
,
Sarah
Williams
,
Evelyn S.
Innes
,
Paige
Barnard
,
Julia
Koehn
,
Somayra S. A.
Mamsa
,
Paul
Quinn
,
Daryl L.
Howard
,
Simon A.
James
,
Mark J.
Hackett
Diamond Proposal Number(s):
[34101]
Open Access
Abstract: Understanding the role of metal ions in normal and abnormal cell function continues to emerge as a critical research area in the biological and biochemical sciences. This is especially true in the context of brain health and neurodegenerative diseases, as the brain is especially enriched in metal ions. A range of microscopy and bioanalytical techniques are available to assist in characterizing and observing changes to the brain metallome. As is the case in many other scientific fields, the integration of multiple analytical methods often yields a more complete chemical picture and deeper biological understanding. Herein, we present a case study applying 4 different analytical methods to provide spatially resolved characterization of chemical and biochemical parameters relating to the iron (Fe) metallome within a specific brain region, cornu ammonis sector 1 (CA1) of the hippocampus. The CA1 hippocampal sector was chosen for investigation due to its known endogenous enrichment in Fe and its selective vulnerability to neurodegeneration. The 4 analytical techniques applied were X-ray fluorescence microscopy (to quantify Fe distribution); X-ray absorption near-edge structure (XANES) spectroscopy to reveal information on Fe oxidation state and coordination environment; immuno-fluorescence to reveal relative abundance of Fe storage proteins (heavy chain ferritin and mitochondrial ferritin); and spatial transcriptomics to reveal gene expression pathways relevant to Fe homeostasis. Collectively, the results highlight that although pyramidal neurons in lateral and medial regions of the hippocampal CA1 sector are morphologically similar, key differences in the Fe metallome are evident. The observed differences within the hippocampal CA1 sector potentially indicate a higher oxidative environment and higher metabolic turnover in medial CA1 neurons relative to lateral CA1 neurons, which may account for the heightened vulnerability to neurodegeneration that is observed in the medial CA1 sector.
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Oct 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[28516]
Abstract: Mutations in the E3 ubiquitin ligase Parkin gene have been linked to early onset Parkinson’s disease. Besides many other roles, Parkin is involved in clearance of damaged mitochondria via mitophagy—a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognizes its substrates for ubiquitination. Here, we characterize a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results may explain fast kinetics of Miro1 ubiquitination by Parkin in recombinant assays and provide a biochemical explanation for Miro1-dependent Parkin recruitment to the mitochondrial membrane observed in cells. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson’s disease.
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Aug 2025
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I03-Macromolecular Crystallography
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Yasushi
Kondo
,
Caitlin
Hatton
,
Robert
Cheng
,
Matilde
Trabuco
,
Hannah
Glover
,
Quentin
Bertrand
,
Fabienne
Stierli
,
Hans-Peter
Seidel
,
Thomas
Mason
,
Sivathmika
Sarma
,
Friedjof
Tellkamp
,
Michal
Kepa
,
Florian
Dworkowski
,
Pedram
Mehrabi
,
Michael
Hennig
,
Joerg
Standfuss
Diamond Proposal Number(s):
[34035]
Open Access
Abstract: Metabotropic glutamate receptor 5 (mGlu5) is implicated in various neurodegenerative disorders, making it an attractive drug target. Although several ligand-bound crystal structures of mGlu5 exist, their apo-state crystal structure remains unknown. Here, we study mGlu5 structural changes using the photochemical affinity switch, alloswitch-1, in combination with time-resolved freeze-trapping methods. By X-ray crystallography, we demonstrated that isomerizing alloswitch-1 leads to its release from the binding pocket within a few seconds. The apo structure, determined at a resolution of 2.9 Å, is more comparable to the inactive state than to the active state. Our approach presents an accessible alternative to time-resolved serial crystallography for capturing thermodynamically stable transient intermediates. The mGlu5 apo-structure provides molecular insights into the ligand-free allosteric pocket, which can guide the design of new allosteric modulators.
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Jul 2025
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I18-Microfocus Spectroscopy
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R.
Kinet
,
M.
Bourdenx
,
S.
Dovero
,
M.
Darricau
,
M.-L.
Arotcarena
,
S.
Camus
,
G.
Porras
,
M.-L.
Thiolat
,
I.
Trigo-Damas
,
S.
Bohic
,
M.
Morari
,
E.
Doudnikoff
,
M.
Goikoetxea
,
S.
Claverol
,
C.
Tokarski
,
N.
Kruse
,
B.
Mollenhauer
,
C.
Estrada
,
N.
Garcia-Carrillo
,
M. T.
Herrero
,
M.
Vila
,
J. A.
Obeso
,
E.
Bezard
,
B.
Dehay
Diamond Proposal Number(s):
[13009]
Open Access
Abstract: The presence of α-synuclein (α-syn) aggregates, such as Lewy bodies in patients with Parkinson’s disease (PD), contributes to dopaminergic cell death. Injection of PD patient–derived α-syn in nonhuman primates has illustrated the exquisite vulnerability of primate dopaminergic neurons. Here, we aimed to elucidate the temporal and spatial pathological changes induced by two distinct α-syn pathogenic structures, having large or small sizes. To unravel the underlying molecular pathways, we conducted a proteomic analysis of the putamen and the entorhinal cortex, two brain regions carrying notable α-syn pathology. We demonstrate that distinct assemblies of α-syn aggregates drive unique pathogenic changes that ultimately result in a comparable extent of nigrostriatal degeneration at the level of nigral dopaminergic neuron cell bodies and striatal dopaminergic terminals. More broadly, our findings identify pathogenic trajectories associated with large or small α-syn aggregates, suggesting the existence of several possible concomitant pathogenic routes in PD.
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Jun 2025
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I04-Macromolecular Crystallography
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Pekka
Kallunki
,
Florence
Sotty
,
Katarina
Willén
,
Michal
Lubas
,
Laurent
David
,
Malene
Ambjørn
,
Ann-Louise
Bergström
,
Louise
Buur
,
Ibrahim
Malik
,
Steffen
Nyegaard
,
Thomas Thiilmark
Eriksen
,
Berit O.
Krogh
,
Jeffrey B.
Stavenhagen
,
Kathrine J.
Andersen
,
Lars Ø.
Pedersen
,
Ersoy
Cholak
,
Edward N.
Van Den Brink
,
Rik
Rademaker
,
Tom
Vink
,
David
Satijn
,
Paul W. H. I.
Parren
,
Søren
Christensen
,
Line R.
Olsen
,
Josefine N.
Søderberg
,
Sandra
Vergo
,
Allan
Jensen
,
Jan
Egebjerg
,
Pernille Gry
Wulff-Larsen
,
Mikkel N.
Harndahl
,
Dina S. M.
Damlund
,
Kaare
Bjerregaard-Andersen
,
Karina
Fog
Open Access
Abstract: Amlenetug (Lu AF82422) is a human monoclonal antibody targeting α-synuclein in clinical development for multiple system atrophy. We describe a series of studies that characterize its functional properties and supported its selection as a viable clinical candidate. Amlenetug inhibits seeding induced in mouse primary neurons by various α-synuclein fibrillar assemblies and by aggregates isolated from MSA brain homogenate. In vivo, both co-injection of amlenetug with α-synuclein assemblies in mouse brain and peripheral administration inhibit α-synuclein seeding. Amlenetug inhibits uptake of α-synuclein seeds as well as accumulation of C-terminal truncated α-synuclein seeds and demonstrates binding to monomeric, aggregated, and truncated forms of human α-synuclein. The epitope of amlenetug was mapped to amino acids 112-117 and further characterized by crystallographic structure analysis. Based on our data, we hypothesize that targeting α-synuclein will potentially slow further disease progression by inhibiting further pathology development but be without impact on established pathology and symptoms.
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May 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[37045]
Open Access
Abstract: Superoxide dismutase 1 (SOD1) is a crucial enzyme that protects cells from oxidative damage by converting superoxide radicals into H2O2 and O2. This detoxification process, essential for cellular homeostasis, relies on a precisely orchestrated catalytic mechanism involving the copper cation, while the zinc cation contributes to the structural integrity of the enzyme. This study presents the 2.3 Å crystal structure of human SOD1 (PDB ID: 9IYK), revealing an assembly of six homodimers and twelve distinct active sites. The water molecules form a complex hydrogen-bonding network that drives proton transfer and sustains active site dynamics. Our structure also uncovers subtle conformational changes that highlight the intrinsic flexibility of SOD1, which is essential for its function. Additionally, we observe how these dynamic structural features may be linked to pathological mutations associated with amyotrophic lateral sclerosis (ALS). By advancing our understanding of hSOD1’s mechanistic intricacies and the influence of water coordination, this study offers valuable insights for developing therapeutic strategies targeting ALS. Our structure’s unique conformations and active site interactions illuminate new facets of hSOD1 function, underscoring the critical role of structural dynamics in enzyme catalysis. Moreover, we conducted a molecular docking analysis using SOD1 for potential radical scavengers and Abelson non-receptor tyrosine kinase (c-Abl, Abl1) inhibitors targeting misfolded SOD1 aggregation along with oxidative stress and apoptosis, respectively. The results showed that CHEMBL1075867, a free radical scavenger derivative, showed the most promising docking results and interactions at the binding site of hSOD1, highlighting its promising role for further studies against SOD1-mediated ALS.
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Apr 2025
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Krios III-Titan Krios III at Diamond
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Diamond Proposal Number(s):
[20287]
Open Access
Abstract: The Hsp70 chaperone system is capable of disassembling pathological aggregates such as amyloid fibres associated with serious degenerative diseases. Here we examine the role of the J-domain protein co-factor in amyloid disaggregation by the Hsc70 system. We used cryo-EM and tomography to compare the assemblies with wild-type DNAJB1 or inactive mutants. We show that DNAJB1 binds regularly along α-synuclein amyloid fibrils and acts in a 2-step recruitment of Hsc70, releasing DNAJB1 auto-inhibition before activating Hsc70 ATPase. The wild-type DNAJB1:Hsc70:Apg2 complex forms dense arrays of chaperones on the fibrils, with Hsc70 on the outer surface. When the auto-inhibition is removed by mutating DNAJB1 (ΔH5 DNAJB1), Hsc70 is recruited to the fibrils at a similar level, but the ΔH5 DNAJB1:Ηsc70:Apg2 complex is inactive, binds less regularly to the fibrils and lacks the ordered clusters. Therefore, we propose that 2-step activation of DNAJB1 regulates the ordered assembly of Hsc70 on the fibril. The localised, dense packing of chaperones could trigger a cascade of recruitment and activation to give coordinated, sequential binding and disaggregation from an exposed fibril end, as previously observed in AFM videos. This mechanism is likely to be important in maintaining a healthy cellular proteome into old age.
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Mar 2025
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B21-High Throughput SAXS
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Open Access
Abstract: The pathological deposition of tau and amyloid-beta into insoluble amyloid fibrils are pathological hallmarks of Alzheimer’s disease. Molecular chaperones are important cellular factors contributing to the regulation of tau misfolding and aggregation. Here we reveal an Hsp90-independent mechanism by which the co-chaperone p23 as well as a molecular complex formed by two co-chaperones, p23 and FKBP51, modulates tau aggregation. Integrating NMR spectroscopy, SAXS, molecular docking, and site-directed mutagenesis we reveal the structural basis of the p23-FKBP51 complex. We show that p23 specifically recognizes the TPR domain of FKBP51 and interacts with tau through its C-terminal disordered tail. We further show that the p23-FKBP51 complex binds tau to form a dynamic p23-FKBP51-tau trimeric complex that delays tau aggregation and thus may counteract Hsp90-FKBP51 mediated toxicity. Taken together, our findings reveal a co-chaperone mediated Hsp90-independent chaperoning of tau protein.
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Jan 2025
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