System-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to... Show moreSystem-level analysis of single-cell data is rapidly transforming the field of immunometabolism. Given the competitive demand for nutrients in immune microenvironments, there is a need to understand how and when immune cells access these nutrients. Here, we describe a new approach for single-cell analysis of nutrient uptake where we use in-cell biorthogonal labeling of a functionalized amino acid after transport into the cell. In this manner, the bona fide active uptake of glutamine via SLC1A5/ASCT2 could be quantified. We used this assay to interrogate the transport capacity of complex immune subpopulations, both in vitro and in vivo. Taken together, our findings provide an easy sensitive single-cell assay to assess which cells support their function via SLC1A5-mediated uptake. This is a significant addition to the single-cell metabolic toolbox required to decode the metabolic landscape of complex immune microenvironments. Show less
The antenna complex of green sulfur bacteria, the chlorosome, is one of the most efficient supramolecular systems for efficient long-range exciton transfer in nature. Femtosecond transient... Show moreThe antenna complex of green sulfur bacteria, the chlorosome, is one of the most efficient supramolecular systems for efficient long-range exciton transfer in nature. Femtosecond transient absorption experiments provide new insight into how vibrationally induced quantum overlap between exciton states supports highly efficient long-range exciton transfer in the chlorosome of Chlorobium tepidum. Our work shows that excitation energy is delocalized over the chlorosome in <1 ps at room temperature. The following exciton transfer to the baseplate occurs in ∼3 to 5 ps, in line with earlier work also performed at room temperature, but significantly faster than at the cryogenic temperatures used in previous studies. This difference can be attributed to the increased vibrational motion at room temperature. We observe a so far unknown impact of the excitation photon energy on the efficiency of this process. This dependency can be assigned to distinct optical domains due to structural disorder, combined with an exciton trapping channel competing with exciton transfer toward the baseplate. An oscillatory transient signal damped in <1 ps has the highest intensity in the case of the most efficient exciton transfer to the baseplate. These results agree well with an earlier computational finding of exciton transfer driven by low-frequency rotational motion of molecules in the chlorosome. Such an exciton transfer process belongs to the quantum coherent regime, for which the Förster theory for intermolecular exciton transfer does not apply. Our work hence strongly indicates that structural flexibility is important for efficient long-range exciton transfer in chlorosomes. Show less
Petrie, G.N.; Balsevich, G.; Fuzesi, T.; Aukema, R.J.; Driever, W.P.F.; Stelt, M. van der; ... ; Hill, M.N. 2023
Background and Purpose: Endocannabinoid (eCB) signalling gates many aspects of the stress response, including the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is controlled by... Show moreBackground and Purpose: Endocannabinoid (eCB) signalling gates many aspects of the stress response, including the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is controlled by corticotropin releasing hormone (CRH) producing neurons in the paraventricular nucleus of the hypothalamus (PVN). Disruption of eCB signalling increases drive to the HPA axis, but the mechanisms subserving this process are poorly understood.Experimental Approach: Using an array of cellular, endocrine and behavioural readouts associated with activation of CRH neurons in the PVN, we evaluated the contributions of tonic eCB signalling to the generation of a stress response.Key Results: The CB1 receptor antagonist/inverse agonist AM251, neutral antagonist NESS243 and NAPE PLD inhibitor LEI401 all uniformly increased Fos in the PVN, unmasked stress-linked behaviours, such as grooming, and increased circulating CORT, recapitulating the effects of stress. Similar effects were also seen after direct administration of AM251 into the PVN, while optogenetic inhibition of PVN CRH neurons ameliorated stress-like behavioural changes produced by disruption of eCB signalling.Conclusions and Implications: These data indicate that under resting conditions, constitutive eCB signalling restricts activation of the HPA axis through local regulation of CRH neurons in the PVN. Show less
To probe the reaction mechanism, underlying the rearrangementofoft-used trichloroacetimidate glycosyl donors into the correspondinganomeric trichloroacetamides, we have used a combination of C-13-... Show moreTo probe the reaction mechanism, underlying the rearrangementofoft-used trichloroacetimidate glycosyl donors into the correspondinganomeric trichloroacetamides, we have used a combination of C-13- and N-15-labeled glycosyl trichloroacetimidate donorsin a series of crossover experiments. These unambiguously show thattrichloroacetamides are formed via an intermolecular aglycon transfermechanism. This insight enables the design of more effective glycosylationprotocols, preventing the formation of dead-end side products. Show less
Gelder, M.A.; Zanden, S.Y. van der; Vriends, M.B.L.; Wagensveld, R.A.; Marel, G.A. van der; Codee, J.D.C.; ... ; Neefjes, J.J.C. 2023
The anthracycline anti-cancer drugs are intensely usedin the clinicto treat a wide variety of cancers. They generate DNA double strandbreaks, but recently the induction of chromatin damage was... Show moreThe anthracycline anti-cancer drugs are intensely usedin the clinicto treat a wide variety of cancers. They generate DNA double strandbreaks, but recently the induction of chromatin damage was introducedas another major determinant of anti-cancer activity. The combinationof these two events results in their reported side effects. Whileour knowledge on the structure-activity relationship of anthracyclineshas improved, many structural variations remain poorly explored. Therefore,we here report on the preparation of a diverse set of anthracyclineswith variations within the sugar moiety, amine alkylation pattern,saccharide chain and aglycone. We assessed the cytotoxicity in vitro in relevant human cancer cell lines, and the capacityto induce DNA- and chromatin damage. This coherent set of data allowedus to deduce a few guidelines on anthracycline design, as well asdiscover novel, highly potent anthracyclines that may be better toleratedby patients. Show less
Activity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity... Show moreActivity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report the use of carmofur- and activity-based probes to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug of 5-fluorouracil and also has antimicrobial and anti-biofilm activity. Carmofur probes were originally designed to target human acid ceramidase, a member of the NTN hydrolase family with an active-site cysteine nucleophile. Here, we first profiled the targets of a fluorescent carmofur probe in live S. aureus under biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatography/mass spectrometry. Treatment with a carmofur-biotin probe led to enrichment of 20 enzymes from diverse families awaiting further characterization, including the NTN hydrolase-related IMP cyclohydrolase PurH. However, the probe preferentially labeled serine hydrolases, thus displaying a reactivity profile similar to that of carbamates. Our results suggest that the electrophilic N-carbamoyl-5-fluorouracil scaffold could potentially be optimized to achieve selectivity towards diverse enzyme families. The observed promiscuous reactivity profile suggests that the clinical use of carmofur presumably leads to inactivation of a number human and microbial enzymes, which could lead to side effects and/or contribute to therapeutic efficacy. Show less
Gracht, D. van der; Rowland, R.J.; Roig-Zamboni, V.; Guimaraes Da Lomba Ferraz, M.J.; Louwerse, G.A.M.; Geurink, P.P.; ... ; Artola Perez de Azanza, M.E. 2023
Lysosomal exoglycosidases are responsible for processing endocytosed glycans from the non-reducing end to produce the corresponding monosaccharides. Genetic mutations in a particular lysosomal... Show moreLysosomal exoglycosidases are responsible for processing endocytosed glycans from the non-reducing end to produce the corresponding monosaccharides. Genetic mutations in a particular lysosomal glycosidase may result in accumulation of its particular substrate, which may cause diverse lysosomal storage disorders. The identification of effective therapeutic modalities to treat these diseases is a major yet poorly realised objective in biomedicine. One common strategy comprises the identification of effective and selective competitive inhibitors that may serve to stabilize the proper folding of the mutated enzyme, either during maturation and trafficking to, or residence in, endo-lysosomal compartments. The discovery of such inhibitors is greatly aided by effective screening assays, the development of which is the focus of the here-presented work. We developed and applied fluorescent activity-based probes reporting on either human GH30 lysosomal glucosylceramidase (GBA1, a retaining & beta;-glucosidase) or GH31 lysosomal retaining & alpha;-glucosidase (GAA). FluoPol-ABPP screening of our in-house 358-member iminosugar library yielded compound classes selective for either of these enzymes. In particular, we identified a class of N-alkyldeoxynojirimycins that inhibit GAA, but not GBA1, and that may form the starting point for the development of pharmacological chaperone therapeutics for the lysosomal glycogen storage disease that results from genetic deficiency in GAA: Pompe disease. Show less
The development of novel anti-infectives requires unprecedented strategies targeting pathways which are solely present in pathogens but absent in humans. Following this principle, we developed... Show moreThe development of novel anti-infectives requires unprecedented strategies targeting pathways which are solely present in pathogens but absent in humans. Following this principle, we developed inhibitors of lipoic acid (LA) salvage, a crucial pathway for the survival of LA auxotrophic bacteria and parasites but non-essential in human cells. An LA-based probe was selectively transferred onto substrate proteins via lipoate protein ligase (LPL) in intact cells, and their binding sites were determined by mass spectrometry. Probe labeling served as a proxy of LPL activity, enabling in situ screenings for cell-permeable LPL inhibitors. Profiling a focused compound library revealed two substrate analogs (LAMe and C3) as inhibitors, which were further validated by binding studies and co-crystallography. Importantly, LAMe exhibited low toxicity in human cells and achieved killing of Plasmodium falciparum in erythrocytes with an EC50 value of 15 μM, making it the most effective LPL inhibitor reported to date. Show less
Eskes, E.C.B; Lienden, M.J.C. van der; Sjouke, B.; Vliet, L. van; Brands, M.M.M.G.; Hollak, C.E.M.; Aerts, J.M.F.G. 2023
Acid sphingomyelinase deficiency (ASMD) is a rare LSD characterized by lysosomal accumulation of sphingomyelin, primarily in macrophages. With the recent availability of enzyme replacement therapy,... Show moreAcid sphingomyelinase deficiency (ASMD) is a rare LSD characterized by lysosomal accumulation of sphingomyelin, primarily in macrophages. With the recent availability of enzyme replacement therapy, the need for biomarkers to assess severity of disease has increased. Glycoprotein non-metastatic protein B (GPNMB) plasma levels were demonstrated to be elevated in Gaucher disease. Given the similarities between Gaucher disease and ASMD, the hypothesis was that GPNMB might be a potential biochemical marker for ASMD as well. Plasma samples of ASMD patients were analyzed and GPNMB plasma levels were compared to those of healthy volunteers. Visceral disease severity was classified as severe when splenic, hepatic and pulmonary manifestations were all present and as mild to moderate if this was not the case. Median GPNMB levels in 67 samples of 19 ASMD patients were 185 ng/ml (range 70-811 ng/ml) and were increased compared to 10 healthy controls (median 36 ng/ml, range 9-175 ng/ml, p < 0.001). Median plasma GPNMB levels of ASMD patients with mild to moderate visceral disease compared to patients with severe visceral disease differed significantly and did not overlap (respectively 109 ng/ml, range 70-304 ng/ml and 325 ng/ml, range 165-811 ng/ml, p < 0.001). Correlations with other biochemical markers of ASMD (i.e. chitotriosidase activity, CCL18 and lysosphingomyelin, respectively R = 0.28, p = 0.270; R = 0.34, p = 0.180; R = 0.39, p = 0.100) and clinical parameters (i.e. spleen volume, liver volume, diffusion capacity and forced vital capacity, respectively R = 0.59, p = 0.061, R = 0.5, p = 0.100, R = 0.065, p = 0.810, R = -0.38, p = 0.160) could not be established within this study. The results of this study suggest that GPNMB might be suitable as a biomarker of visceral disease severity in ASMD. Correlations between GPNMB and biochemical or clinical markers of ASMD and response to therapy have to be studied in a larger cohort. Show less
Catechol-containing natural products are common constituents of foods, drinks, and drugs. Natural products carrying this motif are often associated with beneficial biological effects such as... Show moreCatechol-containing natural products are common constituents of foods, drinks, and drugs. Natural products carrying this motif are often associated with beneficial biological effects such as anticancer activity and neuroprotection. However, the molecular mode of action behind these properties is poorly understood. Here, we apply a mass spectrometry-based competitive chemical proteomics approach to elucidate the target scope of catechol-containing bioactive molecules from diverse foods and drugs. Inspired by the protein reactivity of catecholamine neurotransmitters, we designed and synthesised a broadly reactive minimalist catechol chemical probe based on dopamine. Initial labelling experiments in live human cells demonstrated broad protein binding by the probe, which was largely outcompeted by its parent compound dopamine. Next, we investigated the competition profile of a selection of biologically relevant catechol-containing substances. With this approach, we characterised the protein reactivity and the target scope of dopamine and ten biologically relevant catechols. Strikingly, proteins associated with the endoplasmic reticulum (ER) were among the main targets. ER stress assays in the presence of reactive catechols revealed an activation of the unfolded protein response (UPR). The UPR is highly relevant in oncology and cellular resilience, which may provide an explanation of the health-promoting effects attributed to many catechol-containing natural products. Show less
Correction for 'Automated assessment of redox potentials for dyes in dye-sensitized photoelectrochemical cells' by Jelena Belić et al., Phys. Chem. Chem. Phys., 2022, 24, 197-210, https://doi.org... Show moreCorrection for 'Automated assessment of redox potentials for dyes in dye-sensitized photoelectrochemical cells' by Jelena Belić et al., Phys. Chem. Chem. Phys., 2022, 24, 197-210, https://doi.org/10.1039/D1CP04218A. Show less
Minnee, H.; Chung, H.W.; Rack, J.G.M.; Marel, G.A. van der; Overkleeft, H.S.; Codee, J.D.C.; ... ; Filippov, D.V. 2023
Adenosine diphosphateribosylation (ADP-ribosylation) is a crucialpost-translational modification involved in important regulatory mechanismsof numerous cellular pathways including histone... Show moreAdenosine diphosphateribosylation (ADP-ribosylation) is a crucialpost-translational modification involved in important regulatory mechanismsof numerous cellular pathways including histone maintenance and DNAdamage repair. To study this modification, well-defined ADP-ribosylatedpeptides, proteins, and close analogues thereof have been invaluabletools. Recently, proteomics studies have revealed histidine residuesto be ADP-ribosylated. We describe here the synthesis of a completeset of triazole-isosteres of ADP-ribosylated histidine to serve asprobes for ADP-ribosylating biomachinery. By exploiting Cu(I)- andRu(II)-catalyzed click chemistry between a propargylglycine buildingblock and an & alpha;- or & beta;-configured azidoribose, we have successfullyassembled the & alpha;- and & beta;-configured 1,4- and 1,5-triazoles,mimicking N(& tau;)- and N(& pi;)-ADP-ribosylated histidine, respectively.The ribosylated building blocks could be incorporated into a peptidesequence using standard solid-phase peptide synthesis and transformedon resin into the ADP-ribosylated fragments to provide a total offour ADP-ribosyl triazole conjugates, which were evaluated for theirchemical and enzymatic stability. The 1,5-triazole analogues mimickingthe N(& pi;)-substituted histidines proved susceptible to base-inducedepimerization and the ADP-ribosyl & alpha;-1,5-triazole linkage couldbe cleaved by the (ADP-ribosyl)hydrolase ARH3. Show less
