Ruthenium Phenoxo Complexes: An Isolobal Ligand to Cp with Improved Properties.

Catalytic π-arene activation is based on catalysts that allow for arene exchange. To date, cyclopentadiene (Cp)-derived catalysts are the most commonly used in π-arene activation despite their low arene exchange rates. Herein, we report the synthesis, analysis, and catalytic application of Ru(II) complexes supported by phenoxo ligands, which are isolobal alternatives to Cp. The phenoxo complexes exhibit arene exchange rates significantly faster than those of the corresponding Cp complexes. The rate can be further increased through the choice of appropriate counterions. The mechanism of the arene exchange process is elucidated by kinetic and computational analyses. We demonstrate the utility of the new catalysts through an SNAr reaction between fluorobenzene and alcohols, including secondary alcohols that could not be used previously in related reactions. Moreover, the catalytic thermal decarboxylation of phenylacetic acids is presented.

pH-Controlled Intramolecular Decarboxylative Cyclization of Biarylacetic Acids: Implication on Umpolung Reactivity of Aroyl Radicals.

A simple approach for the intramolecular aroylation of electron-rich arenes under mild conditions has been developed. A pH-controlled polarity umpolung strategy can be used to synthesize different fluorenones, which are important building blocks for biological applications. Unlike previous acylation reactions involving nucleophilic aroyl radicals, this approach likely relies on in situ generated electrophilic aroyl radicals. Detailed mechanistic and computational investigations provide detailed insights into the reaction mechanism and support the hypothesis of a pH-mediated umpolung.

Effect of Triple Combination CFTR Modulator Therapy on Sleep in Adult Patients with Cystic Fibrosis.

BACKGROUND: Sleep-disordered breathing (SDB) and disturbed sleep are common, often underrecognized, comorbidities in people with cystic fibrosis (pwCF). OBJECTIVES: We studied the effect of CFTR triple combination therapy elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) on sleep in pwCF. METHOD: This was a prospective, observational sleep study in clinically stable adult pwCF. All participants underwent overnight polysomnography (PSG), before (T0) and after (T1) initiation of CFTR modulator therapy with ELX/TEZ/IVA. In addition, pulmonary function tests, calculation of BMI, and sweat chloride testing were performed. RESULTS: Twenty-nine pwCF (mean age 32 ± 8 years; 15 female) participated in the study. Mean time between T0 and T1 was 194 ± 21 days. Total sleep time (TST) was 298 ± 40 min, with decreased sleep efficiency (SE) (76 ± 109) and increased sleep latency (SL) (73 ± 38 min). Sleep stages for NREM (N1-3) and REM sleep were within the normal range. Nocturnal respiratory events mainly occur during REM sleep (T0: AHI REM 8.3 ± 9.0/h; ODI REM 9.4 ± 10.6/h), whereas the overall AHI was normal (3.6 ± 3.7/h). After initiation of ELX/TEZ/IVA, we saw significant improvements in ppFEV1 (p < 0.001) and BMI (p < 0.001) and a reduction in sweat chloride levels (p < 0.001). In parallel, there was a reduction in AHI (p = 0.003), ODI (p = 0.001), and nocturnal respiratory rate (p < 0.001), both in total, REM and NREM sleep. Neither TST, SL, SE, nor sleep architecture was influenced (all p > 0.05). CONCLUSIONS: Initiation of ELX/TEZ/IVA resulted in significant improvements in SDB in adult pwCF.

Obstructive sleep apnea and nocturnal hypoxemia in adult patients with cystic fibrosis.

BACKGROUND: Obstructive sleep apnea (OSA), nocturnal hypoxemia and excessive daytime sleepiness (EDS) are common comorbidities in people with cystic fibrosis (pwCF). Most of the data showing this originates from children and adolescents. The aim of this study was to collect data on sleep parameters, EDS and pulmonary function from a large cohort of adult pwCF. METHODS: Full overnight polysomnography (PSG) was performed. EDS was determined using the Epworth Sleepiness Scale (ESS). Demographic and clinical data (body mass index [BMI], pulmonary function, capillary blood gases) were collected. RESULTS: A total of 52 adult pwCF were included (mean age 30.7 ± 8.0 years, mean percent predicted forced expiratory volume in 1 s [ppFEV1] of 52.1 ± 14.8). Overall AHI was in the normal range (4.5 ± 4.0/h); 21/52 pwCF (40%) had an apnea-hypopnea index > 5/h. Nocturnal hypoxemia was found in 25% of participants and this was associated with ppFEV1 (p = 0.014), awake oxygen saturation (SpO2; p = 0.021) and awake partial pressure of oxygen (pO2; p = 0.003); there were no significant differences in age, lung function and BMI were found for pwCF with versus without OSA (all p > 0.05). Eight pwCF (15%) had an ESS score > 10 (indicating EDS). OSA was best predicted by awake pO2 (area under the curve [AUC] 0.66, p = 0.048), while nocturnal hypoxemia was best predicted by ppFEV1 (AUC 0.74, p = 0.009), awake pO2 (AUC 0.76, p = 0.006) and awake SpO2 (AUC 0.71; p = 0.025). CONCLUSION: OSA, nocturnal hypoxemia and EDS were common in adult pwCF, but no strong predictors were identified. Therefore, we suggest regular PSG and ESS scoring in adult pwCF, regardless of disease severity.

Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains.

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

Amide Synthesis by Nickel/Photoredox-Catalyzed Direct Carbamoylation of (Hetero)Aryl Bromides.

Herein, we report a one-electron strategy for catalytic amide synthesis that enables the direct carbamoylation of (hetero)aryl bromides. This radical cross-coupling approach, which is based on the combination of nickel and photoredox catalysis, proceeds at ambient temperature and uses readily available dihydropyridines as precursors of carbamoyl radicals. The method's mild reaction conditions make it tolerant of sensitive-functional-group-containing substrates and allow the installation of an amide scaffold within biologically relevant heterocycles. In addition, we installed amide functionalities bearing electron-poor and sterically hindered amine moieties, which would be difficult to prepare with classical dehydrative condensation methods.

Changing the site energy of per-614 in the Peridinin-chlorophyll a-protein does not alter its capability of chlorophyll triplet quenching.

The peridinin-chlorophyll-a protein (PCP) is a water-soluble light harvesting protein of the dinoflagellate Amphidinium carterae, employing peridinin (Per) as the main carotenoid to fulfil light harvesting and photo-protective functions. Per molecules bound to the protein experience specific molecular surroundings which lead to different electronic and spectral properties. In the refolded N89 L variant PCP (N89 L-RFPCP) a significant part of the intensity on the long wavelength side of the absorption spectrum is shifted to shorter wavelengths due to a significant change in the Per-614 site energy. Since Per-614 has been shown to be the main chlorophyll (Chl) triplet quencher in the protein, and the relative geometry of pigments is not affected by the mutation as verified by X-ray crystallography, this variant is ideally suited to study the dependence of the triplet-triplet energy transfer (TTET) mechanism on the pigment site energy. By using a combination of Optically Detected Magnetic Resonance (ODMR), pulse Electron Paramagnetic Resonance (EPR) and Electron Nuclear DOuble Resonance (ENDOR) we found that PCP maintains the efficient Per-614-to-Chl-a TTET despite the change of Per-614 local energy. This shows the robustness of the photoprotective site, which is very important for the protection of the system.

Detecting ligand interactions in real time on living bacterial cells.

Time-resolved analysis assays of receptor-ligand interactions are fundamental in basic research and drug discovery. Adequate methods are well developed for the analysis of recombinant proteins such as antibody-antigen interactions. However, assays for time-resolved ligand-binding processes on living cells are still rare, in particular within microbiology. In this report, the real-time cell-binding assay (RT-CBA) technology LigandTracer®, originally designed for mammalian cell culture, was extended to cover Gram-positive and Gram-negative bacteria. This required the development of new immobilization methods for bacteria, since LigandTracer depends on cells being firmly attached to a Petri dish. The evaluated Escherichia coli CJ236 and BL21 as well as Staphylococcus carnosus TM300 strains were immobilized to plastic Petri dishes using antibody capture, allowing us to depict kinetic binding traces of fluorescently labeled antibodies directed against surface-displayed bacterial proteins for as long as 10-15 h. Interaction parameters, such as the affinity and kinetic constants, could be estimated with high precision (coefficient of variation 9-44%) and the bacteria stayed viable for at least 16 h. The other tested attachment protocols were inferior to the antibody capture approach. Our attachment protocol is generic and could potentially also be applied to other assays and purposes.

Distance measurements in peridinin-chlorophyll a-protein by light-induced PELDOR spectroscopy. Analysis of triplet state localization.

Triplet-triplet energy transfer from chlorophylls to carotenoids is the mechanism underlying the photoprotective role played by carotenoids in many light harvesting complexes, during photosynthesis. The peridinin-chlorophyll-a protein (PCP) is a water-soluble light harvesting protein of the dinoflagellate Amphidinium carterae, employing peridinin as the main carotenoid to fulfil this function. The dipolar coupling of the triplet state of peridinin, populated under light excitation in isolated PCP, to the MTSSL nitroxide, introduced in the protein by site-directed mutagenesis followed by spin labeling, has been measured by Pulse ELectron-electron DOuble Resonance (PELDOR) spectroscopy. The triplet-nitroxide distance derived by this kind of experiments, performed for the first time in a protein system, allowed the assignment of the triplet state to a specific peridinin molecule belonging to the pigment cluster. The analysis strongly suggests that this peridinin is the one in close contact with the water ligand to the chlorophyll a, thus supporting previous evidences based on ENDOR and time resolved-EPR.

Viral and cellular proteins containing FGDF motifs bind G3BP to block stress granule formation.

The Ras-GAP SH3 domain-binding proteins (G3BP) are essential regulators of the formation of stress granules (SG), cytosolic aggregates of proteins and RNA that are induced upon cellular stress, such as virus infection. Many viruses, including Semliki Forest virus (SFV), block SG induction by targeting G3BP. In this work, we demonstrate that the G3BP-binding motif of SFV nsP3 consists of two FGDF motifs, in which both phenylalanine and the glycine residue are essential for binding. In addition, we show that binding of the cellular G3BP-binding partner USP10 is also mediated by an FGDF motif. Overexpression of wt USP10, but not a mutant lacking the FGDF-motif, blocks SG assembly. Further, we identified FGDF-mediated G3BP binding site in herpes simplex virus (HSV) protein ICP8, and show that ICP8 binding to G3BP also inhibits SG formation, which is a novel function of HSV ICP8. We present a model of the three-dimensional structure of G3BP bound to an FGDF-containing peptide, likely representing a binding mode shared by many proteins to target G3BP.

Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14-3-3 protein.

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon infection, parasitized dendritic cells (DCs) and microglia exhibit a hypermigratory phenotype in vitro that has been associated with enhancing parasite dissemination in vivo in mice. One unresolved question is how parasites commandeer parasitized cells to achieve systemic dissemination by a 'Trojan-horse' mechanism. By chromatography and mass spectrometry analyses, we identified an orthologue of the 14-3-3 protein family, T. gondii 14-3-3 (Tg14-3-3), as mediator of DC hypermotility. We demonstrate that parasite-derived polypeptide fractions enriched for Tg14-3-3 or recombinant Tg14-3-3 are sufficient to induce the hypermotile phenotype when introduced by protein transfection into murine DCs, human DCs or microglia. Further, gene transfer of Tg14-3-3 by lentiviral transduction induced hypermotility in primary human DCs. In parasites expressing Tg14-3-3 in a ligand-regulatable fashion, overexpression of Tg14-3-3 was correlated with induction of hypermotility in parasitized DCs. Localization studies in infected DCs identified Tg14-3-3 within the parasitophorous vacuolar space and a rapid recruitment of host cell 14-3-3 to the parasitophorous vacuole membrane. The present work identifies a determinant role for Tg14-3-3 in the induction of the migratory activation of immune cells by T. gondii. Collectively, the findings reveal Tg14-3-3 as a novel target for an intracellular pathogen that acts by hijacking the host cell's migratory properties to disseminate.

Nitrate reduction enables safer aryldiazonium chemistry.

Aryldiazonium salts remain a staple in organic synthesis and are still prepared largely in accord with the protocol developed in the 19th century. Because of the favorable reactivity that often cannot be achieved with other aryl(pseudo)halides, diazonium chemistry continues to grow. Facile extrusion of dinitrogen contributes to the desired reactivity but is also reason for safety concerns. Explosions have occurred since the discovery of these reagents and still result in accidents. In this study, we report a diazonium chemistry paradigm shift based on nitrate reduction using thiosulfate or dihalocuprates as electron donors that avoids diazonium accumulation. Because nitrate reduction is rate-limiting, aryldiazoniums are produced as fleeting intermediates, which results in a safer and often more efficient deaminative halogenation in a single step from anilines.

The acidic intrinsically disordered region of the inflammatory mediator HMGB1 mediates fuzzy interactions with CXCL12.

Chemokine heterodimers activate or dampen their cognate receptors during inflammation. The CXCL12 chemokine forms with the fully reduced (fr) alarmin HMGB1 a physiologically relevant heterocomplex (frHMGB1•CXCL12) that synergically promotes the inflammatory response elicited by the G-protein coupled receptor CXCR4. The molecular details of complex formation were still elusive. Here we show by an integrated structural approach that frHMGB1•CXCL12 is a fuzzy heterocomplex. Unlike previous assumptions, frHMGB1 and CXCL12 form a dynamic equimolar assembly, with structured and unstructured frHMGB1 regions recognizing the CXCL12 dimerization surface. We uncover an unexpected role of the acidic intrinsically disordered region (IDR) of HMGB1 in heterocomplex formation and its binding to CXCR4 on the cell surface. Our work shows that the interaction of frHMGB1 with CXCL12 diverges from the classical rigid heterophilic chemokines dimerization. Simultaneous interference with multiple interactions within frHMGB1•CXCL12 might offer pharmacological strategies against inflammatory conditions.

The Cryo-EM STRUCTURE of Renal Amyloid Fibril Suggests Structurally Homogeneous Multiorgan Aggregation in AL Amyloidosis.

Immunoglobulin light chain amyloidosis (AL) is caused by the aberrant production of amyloidogenic light chains (LC) that accumulate as amyloid deposits in vital organs. Distinct LC sequences in each patient yield distinct amyloid structures. However different tissue microenvironments may also cause identical protein precursors to adopt distinct amyloid structures. To address the impact of the tissue environment on the structural polymorphism of amyloids, we extracted fibrils from the kidney of an AL patient (AL55) whose cardiac amyloid structure was previously determined by our group. Here we show that the 4.0 Å resolution cryo-EM structure of the renal fibril is virtually identical to that reported for the cardiac fibril. These results provide the first structural evidence that LC amyloids independently deposited in different organs of the same AL patient share a common fold.

Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation.

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH.

G3BP is the central node within stress-induced protein-RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.

AA-amyloidosis in cats (Felis catus) housed in shelters.

Systemic AA-amyloidosis is a protein-misfolding disease characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. A high prevalence of AA-amyloidosis has been reported in captive cheetahs and a horizontal transmission has been proposed. In domestic cats, AA-amyloidosis has been mainly described in predisposed breeds but only rarely reported in domestic short-hair cats. Aims of the study were to determine AA-amyloidosis prevalence in dead shelter cats. Liver, kidney, spleen and bile were collected at death in cats from 3 shelters. AA-amyloidosis was scored. Shedding of amyloid fibrils was investigated with western blot in bile and scored. Descriptive statistics were calculated. In the three shelters investigated, prevalence of AA-amyloidosis was 57.1% (16/28 cats), 73.0% (19/26) and 52.0% (13/25), respectively. In 72.9% of cats (35 in total) three organs were affected concurrently. Histopathology and immunofluorescence of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B = 0.026, CI95% = 0.007-0.046; p = 0.010). AA-amyloidosis was very frequent in shelter cats. Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease. In conclusion, AA-amyloidosis was very frequent in shelter cats and those staying longer had more deposits. The cat may represent a natural model of AA-amyloidosis.

Identification of a single peridinin sensing Chl-a excitation in reconstituted PCP by crystallography and spectroscopy.

The peridinin-chlorophyll a-protein (PCP) of dinoflagellates is unique among the large variety of natural photosynthetic light-harvesting systems. In contrast to other chlorophyll protein complexes, the soluble PCP is located in the thylakoid lumen, and the carotenoid pigments outnumber the chlorophylls. The structure of the PCP complex consists of two symmetric domains, each with a central chlorophyll a (Chl-a) surrounded by four peridinin molecules. The protein provides distinctive surroundings for the pigment molecules, and in PCP, the specific environment around each peridinin results in overlapping spectral line shapes, suggestive of different functions within the protein. One particular Per, Per-614, is hypothesized to show the strongest electronic interaction with the central Chl-a. We have performed an in vitro reconstitution of pigments into recombinant PCP apo-protein (RFPCP) and into a mutated protein with an altered environment near Per-614. Steady-state and transient optical spectroscopic experiments comparing the RFPCP complex with the reconstituted mutant protein identify specific amino acid-induced spectral shifts. The spectroscopic assignments are reinforced by a determination of the structures of both RFPCP and the mutant by x-ray crystallography to a resolution better than 1.5 A. RFPCP and mutated RFPCP are unique in representing crystal structures of in vitro reconstituted light-harvesting pigment-protein complexes.

Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter.

AA amyloidosis is a systemic disease characterized by deposition of misfolded serum amyloid A protein (SAA) into cross-ß amyloid in multiple organs in humans and animals. AA amyloidosis occurs at high SAA serum levels during chronic inflammation. Prion-like transmission was reported as possible cause of extreme AA amyloidosis prevalence in captive animals, e.g. 70% in cheetah and 57-73% in domestic short hair (DSH) cats kept in zoos and shelters, respectively. Herein, we present the 3.3 Å cryo-EM structure of AA amyloid extracted post-mortem from the kidney of a DSH cat with renal failure, deceased in a shelter with extreme disease prevalence. The structure reveals a cross-ß architecture assembled from two 76-residue long proto-filaments. Despite >70% sequence homology to mouse and human SAA, the cat SAA variant adopts a distinct amyloid fold. Inclusion of an eight-residue insert unique to feline SAA contributes to increased amyloid stability. The presented feline AA amyloid structure is fully compatible with the 99% identical amino acid sequence of amyloid fragments of captive cheetah.

Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal serine-rich repeat protein (PsrP-BR) reveal a rigid monomer in solution.

The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a suggested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended ß-sheet on one side, and another ß-sheet that is distorted by loops and ß-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular ß-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation during biofilm formation. Here we report the near complete 15N/13C/1H backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic 15N-T1, T2 and NOE data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding.