Light upconversion is a very powerful tool in bioimaging as it can eliminate autofluorescence, increase imaging contrast, reduce irradiation damage, and increase excitation penetration depth in vivo. ...In particular, triplet–triplet annihilation upconverting (TTA‐UC) nanoparticles and liposomes offer high upconversion efficiency at low excitation power. However, TTA‐UC is quenched in air by oxygen, which also leads to the formation of toxic singlet oxygen. In this work, polyisobutylene‐monomethyl polyethylene glycol block copolymers are synthesized and used for preparing polymersomes that upconvert red light into blue light in absence of oxygen. In addition, it is demonstrated that biocompatible antioxidants such as l‐ascorbate, glutathionate, l‐histidine, sulfite, trolox, or even opti‐MEM medium, can be used to protect the TTA‐UC process in these polymersomes resulting in red‐to‐blue upconversion under aerobic conditions. Most importantly, this approach is also functional in living cells. When A549 lung carcinoma cells are treated with TTA‐UC polymersomes in the presence of 5 × 10−3
m ascorbate and glutathionate, upconversion in the living cells is one order of magnitude brighter than that observed without antioxidants. These results propose a simple chemical solution to the issue of oxygen sensitivity of TTA‐UC, which is of paramount importance for the technological advancement of this technique in biology.
Oxygen sensitivity is a major obstacle for the biological applications of triplet‐triplet annihilation upconverting nanosystems. It is demonstrated that upconverting polymersomes can emit stable upconverted light in air by simply adding low amounts of biological anti‐oxidants. This strategy also works in living cancer cells, where 10× more upconversion occurred when the polymersomes were co‐incubated in presence of antioxidants.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein, and LRRK2 mutants are recognized risk factors for Parkinson’s disease (PD). Although the precise mechanisms that control LRRK2 ...regulation and function are unclear, the importance of the kinase domain is strongly implicated, since 2 of the 5 most common familial LRRK2 mutations (G2019S and I2020T) are localized to the conserved DFGψ motif in the kinase core, and kinase inhibitors are under development. Combining the concept of regulatory (R) and catalytic (C) spines with kinetic and cell-based assays, we discovered a major regulatory mechanism embedded within the kinase domain and show that the DFG motif serves as a conformational switch that drives LRRK2 activation. LRRK2 is quite unusual in that the highly conserved Phe in the DFGψ motif, which is 1 of the 4 R-spine residues, is replaced with tyrosine (DY2018GI). A Y2018F mutation creates a hyperactive phenotype similar to the familial mutation G2019S. The hydroxyl moiety of Y2018 thus serves as a “brake” that stabilizes an inactive conformation; simply removing it destroys a key hydrogen-bonding node. Y2018F, like the pathogenic mutant I2020T, spontaneously forms LRRK2-decorated microtubules in cells, while the wild type and G2019S require kinase inhibitors to form filaments. We also explored 3 different mechanisms that create kinase-dead pseudokinases, including D2017A, which further emphasizes the highly synergistic role of key hydrophobic and hydrophilic/charged residues in the assembly of active LRRK2. We thus hypothesize that LRRK2 harbors a classical protein kinase switch mechanism that drives the dynamic activation of full-length LRRK2.
Red-to-blue triplet-triplet annihilation upconversion was obtained in giant unilamellar vesicles. The upconverted light was homogeneously distributed across the membrane and could be utilized for the ...imaging of individual giant vesicles in three dimensions. These results show the great potential of TTA-UC for imaging applications under anoxic conditions.
The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease-related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass ...spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a "cap" that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the ...more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.
Proteins of the 14-3-3 family are well known modulators of the leucine-rich repeat kinase 2 (LRRK2) regulating kinase activity, cellular localization, and ubiquitylation. Although binding between ...those proteins has been investigated, a comparative study of all human 14-3-3 isoforms interacting with LRRK2 is lacking so far. In a comprehensive approach, we quantitatively analyzed the interaction between the seven human 14-3-3 isoforms and LRRK2-derived peptides covering both, reported and putative 14-3-3 binding sites. We observed that phosphorylation is an absolute prerequisite for 14-3-3 binding and generated binding patterns of 14-3-3 isoforms to interact with peptides derived from the N-terminal phosphorylation cluster (S910 and S935), the Roc domain (S1444) and the C-terminus. The tested 14-3-3 binding sites in LRRK2 preferentially were recognized by the isoforms γ and η, whereas the isoforms ϵ and especially σ showed the weakest or no binding. Interestingly, the possible pathogenic mutation Q930R in LRRK2 drastically increases binding affinity to a peptide encompassing pS935. We then identified the autophosphorylation site T2524 as a so far not described 14-3-3 binding site at the very C-terminus of LRRK2. Binding affinities of all seven 14-3-3 isoforms were quantified for all three binding regions with pS1444 displaying the highest affinity of all measured singly phosphorylated peptides. The strongest binding was detected for the combined phosphosites S910 and S935, suggesting that avidity effects are important for high affinity interaction between 14-3-3 proteins and LRRK2.
To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered ...approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2
retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2
and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.
Despite remarkable progress in applied Surface Plasmon Resonance (SPR)-based methods, concise monitoring of kinetic properties for native biomarkers from patient samples is still lacking. Not only ...are low concentrations of native targets in patient samples, often in the pM range, a limiting and challenging factor, but body fluids as complex matrices furthermore complicate measurements. The here-described method enables the determination of kinetic constants and resulting affinities for native antigens from patients' cerebrospinal fluid (CSF) and sera binding to antibodies. Using a significantly extended target-enrichment step, we modified a common sandwich-assay protocol, based on a primary and secondary antibody. We successfully analyze antibody kinetics of native targets from a variety of origins, with consistent results, independent of their source. Moreover, native neurofilament light chain (NFL) was investigated as an exemplary biomarker. Obtained data reveal antibodies recognizing recombinant NFL with high affinities, while showing no, or only significantly weakened binding to native NFL. The indicated differences for recombinant vs. native material demonstrate another beneficial application. Our assay is highly suitable for gaining valuable insights into characteristics of native biomarkers, thus impacting on the binder development of diagnostic reagents or pharmaceutical drugs.
Protein kinases and GTPases are the two major molecular switches that regulate much of biology, and both of these domains are embedded within the large multi-domain Leucine-Rich Repeat Kinase 2 ...(LRRK2). Mutations in LRRK2 are the most common cause of familial Parkinson’s Disease (PD) and are also implicated in Crohn’s disease (Hui et al., 2018). The recent Cryo Electron Microscopy (Cryo-EM) structure of the four C-terminal domains (RCKW) of LRRK2 includes both of the catalytic domains (Deniston et al., 2020). Although the important allosteric N-terminal domains are missing in the Cryo-EM structure this structure allows us to not only explore the conserved features of the kinase domain, which is trapped in an inactive and open conformation, but also to observe the direct allosteric cross talk between the two domains. To define the unique features of the kinase domain and to better understand the dynamic switch mechanism that allows LRRK2 to toggle between its inactive and active conformations, we have compared the LRRK2 kinase domain to Src, BRaf, and PKA. We also compare and contrast the two canonical glycine-rich loop motifs in LRRK2 that anchor the nucleotide: the G-Loop in protein kinases that anchors ATP and the P-Loop in GTPases that anchors GTP. The GTPase domain of LRRK2 belongs to the Roco protein family and plays an important role as an allosteric effector domain (Bosgraaf et al., 2003, Marin et al., 2008). The RCKW structure also provides a template for the cross-talk between the kinase and GTPase domains and brings new mechanistic insights into the physiological function of LRRK2 and how the kinase domain, along with key phosphorylation sites, can serve as an allosteric hub for mediating conformational changes.
In this cross-sectional study, we investigated whether there is evidence for hepatic mitochondrial dysfunction in manifest and/or premanifest Huntington disease (HD) by using the ¹³C-methionine ...breath test.
The ¹³C-methionine breath test was performed within a group of 21 patients with early manifest HD without medication, 30 premanifest mutation carriers, as well as 36 healthy controls. Premanifest mutation carriers were stratified into the 2 groups preHD-A (further from predicted onset) and preHD-B (nearer) based on a calculation of the probability of estimated disease onset within 5 years. The ¹³C-methionine breath test was performed after an overnight fasting, breath samples were analyzed by nondispersive isotope-selective infrared spectroscopy, and results expressed as percentage dose recovered after 90 minutes of testing time. Statistical analyses comprised analysis of covariance and post hoc t tests.
Patients with manifest HD and mutation carriers from our preHD-B group revealed a lower amount of exhaled ¹³CO₂ compared with healthy controls (p < 0.001 and p = 0.017, respectively). In a stepwise linear regression model, breath test results correlate to functional and cognitive scores of the Unified Huntington's Disease Rating Scale in manifest and also in premanifest HD. For all mutation carriers together, there was a weak but significant correlation of breath test results to ratio caudate volume/total intracranial volume.
This study demonstrates for the first time in vivo a subclinical, hepatic involvement in manifest and premanifest HD.
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