Tethering Cells via Enzymatic Oxidative Crosslinking Enables Mechanotransduction in Non-Cell-Adhesive Materials

Cell–matrix interactions govern cell behavior and tissue function by facilitating transduction of biomechanical cues. Engineered tissues often incorporate these interactions by employing cell-adhesive materials. However, using constitutively active cell-adhesive materials impedes control over cell fate and elicits inflammatory responses upon implantation. Here, an alternative cell–material interaction strategy that provides mechanotransducive properties via discrete inducible on-cell crosslinking (DOCKING) of materials, including those that are inherently non-cell-adhesive, is introduced. Specifically, tyramine-functionalized materials are tethered to tyrosines that are naturally present in extracellular protein domains via enzyme-mediated oxidative crosslinking. Temporal control over the stiffness of on-cell tethered 3D microniches reveals that DOCKING uniquely enables lineage programming of stem cells by targeting adhesome-related mechanotransduction pathways acting independently of cell volume changes and spreading. In short, DOCKING represents a bioinspired and cytocompatible cell-tethering strategy that offers new routes to study and engineer cell–material interactions, thereby advancing applications ranging from drug delivery, to cell-based therapy, and cultured meat

Dietary Sargassum fusiforme improves memory and reduces amyloid plaque load in an Alzheimer's disease mouse model

Activation of liver X receptors (LXRs) by synthetic agonists was found to improve cognition in Alzheimer's disease (AD) mice. However, these LXR agonists induce hypertriglyceridemia and hepatic steatosis, hampering their use in the clinic. We hypothesized that phytosterols as LXR agonists enhance cognition in AD without affecting plasma and hepatic triglycerides. Phytosterols previously reported to activate LXRs were tested in a luciferase-based LXR reporter assay. Using this assay, we found that phytosterols commonly present in a Western type diet in physiological concentrations do not activate LXRs. However, a lipid extract of the 24(S)-Saringosterol-containing seaweed Sargassum fusiforme did potently activate LXR beta. Dietary supplementation of crude Sargassum fusiforme or a Sargassum fusiforme-derived lipid extract to AD mice significantly improved short-term memory and reduced hippocampal A beta plaque load by 81%. Notably, none of the side effects typically induced by full synthetic LXR agonists were observed. In contrast, administration of the synthetic LXRa activator, AZ876, did not improve cognition and resulted in the accumulation of lipid droplets in the liver. Administration of Sargassum fusiforme-derived 24(S)-Saringosterol to cultured neurons reduced the secretion of A beta 42. Moreover, conditioned medium from 24(S)-Saringosterol-treated astrocytes added to microglia increased phagocytosis of A beta. Our data show that Sargassum fusiforme improves cognition and alleviates AD pathology. This may be explained at least partly by 24(S)-Saringosterol-mediated LXR beta activation.</p

An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation

To characterize molecular changes during cell type transitions, the authors develop a method to simultaneously measure protein expression and thermal stability changes. They apply this approach to study differences between human pluripotent stem cells, their progenies, parental and allogeneic cells. Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro

Diving into cellular signalling : Functional proteome analysis by mass spectrometry based approaches

The main aim of my work as described in this thesis has been to improve on proteomics technologies to study protein phosphorylation, protein oxidation and interactions between proteins and drug molecules. In chapter two, we were the first to perform thermal proteome profiling on zebrafish lysate. Thermal proteome profiling is a valuable technique in which mass spectrometry is used to find the on- and off-targets of small ligands, such as drugs. By finding the toxic off-targets of drugs, their development may be improved. Traditionally, thermal proteome profiling experiments were performed on single cell types, causing information on tissue specific proteins to be lost. Here, we improved on this methodology by performing thermal proteome profiling on zebrafish embryo lysate, which harbors all tissue specific proteins. We first showed, as a proof of principle, that we could detect ligand induced stability changes in pervanadate treated lysate, after which we extended this to the selective STAT3 inhibitor napabucasin. Using our approach, we validated the mode of action of napabucasin, while simultaneously finding aldehyde dehydrogenases as off-targets. In chapter three, we investigated the labile post-translational modification phosphohistidine. Phosphohistidine has been very difficult to study in the past, due to not being compatible with standard enrichment strategies. However, recently a novel approach has been developed which allows the identification of this PTM on a proteome wide scale. It was shown that phosphohistidine plays an important role in bacteria such as E.coli, however the importance and scope of this PTM in mammalian systems is not known. Here, we investigated the extent of phosphohistidine in mammalian cells using this optimized workflow. Many novel sites were found, but the validity of these was questioned. Therefore, acidification of the samples was used as a negative control. In E.coli, this drastically decreased the presence of phosphohistidine, while in mammalian samples this behavior was not replicated. Therefore, we concluded that the sites found in our experiments are false positives, and that the contribution of phosphohistidine in mammalian systems is extremely limited. In chapter four, we investigated the oxidative behavior of the catalytic cysteine of the tyrosine phosphatase SHP2 and its mutants. The oxidation of the catalytic cysteine of SHP2 is a known mechanism to (ir)reversibly inactivate it, but we were curious how the rates of oxidation differ between the wildtype phosphatase and the catalytically more active Noonan mutant. This mutant is in a more open conformation compared to the wildtype, which might cause it to be more readily oxidized. Indeed, through a differential alkylation approach we showed that the Noonan mutant is more readily oxidized compared to the wildtype. Additionally, we showed that the addition of catalase to SHP2 in a fusion protein can efficiently protect the catalytic cysteine against hydrogen peroxide. In the future, these fusion proteins may be used to determine the oxidation status of SHP2 in vivo. Lastly, in chapter five I share my view on the future of proteomics. In addition, a lay summary of this thesis and my acknowledgements can be found here

Diving into cellular signalling : Functional proteome analysis by mass spectrometry based approaches

The main aim of my work as described in this thesis has been to improve on proteomics technologies to study protein phosphorylation, protein oxidation and interactions between proteins and drug molecules. In chapter two, we were the first to perform thermal proteome profiling on zebrafish lysate. Thermal proteome profiling is a valuable technique in which mass spectrometry is used to find the on- and off-targets of small ligands, such as drugs. By finding the toxic off-targets of drugs, their development may be improved. Traditionally, thermal proteome profiling experiments were performed on single cell types, causing information on tissue specific proteins to be lost. Here, we improved on this methodology by performing thermal proteome profiling on zebrafish embryo lysate, which harbors all tissue specific proteins. We first showed, as a proof of principle, that we could detect ligand induced stability changes in pervanadate treated lysate, after which we extended this to the selective STAT3 inhibitor napabucasin. Using our approach, we validated the mode of action of napabucasin, while simultaneously finding aldehyde dehydrogenases as off-targets. In chapter three, we investigated the labile post-translational modification phosphohistidine. Phosphohistidine has been very difficult to study in the past, due to not being compatible with standard enrichment strategies. However, recently a novel approach has been developed which allows the identification of this PTM on a proteome wide scale. It was shown that phosphohistidine plays an important role in bacteria such as E.coli, however the importance and scope of this PTM in mammalian systems is not known. Here, we investigated the extent of phosphohistidine in mammalian cells using this optimized workflow. Many novel sites were found, but the validity of these was questioned. Therefore, acidification of the samples was used as a negative control. In E.coli, this drastically decreased the presence of phosphohistidine, while in mammalian samples this behavior was not replicated. Therefore, we concluded that the sites found in our experiments are false positives, and that the contribution of phosphohistidine in mammalian systems is extremely limited. In chapter four, we investigated the oxidative behavior of the catalytic cysteine of the tyrosine phosphatase SHP2 and its mutants. The oxidation of the catalytic cysteine of SHP2 is a known mechanism to (ir)reversibly inactivate it, but we were curious how the rates of oxidation differ between the wildtype phosphatase and the catalytically more active Noonan mutant. This mutant is in a more open conformation compared to the wildtype, which might cause it to be more readily oxidized. Indeed, through a differential alkylation approach we showed that the Noonan mutant is more readily oxidized compared to the wildtype. Additionally, we showed that the addition of catalase to SHP2 in a fusion protein can efficiently protect the catalytic cysteine against hydrogen peroxide. In the future, these fusion proteins may be used to determine the oxidation status of SHP2 in vivo. Lastly, in chapter five I share my view on the future of proteomics. In addition, a lay summary of this thesis and my acknowledgements can be found here

Proteomic tools to study drug function

With the ever growing repertoire of drugs being developed, new unbiased methods are urgently needed that allow fast screening of protein targets and off-targets. Ideally, these methods are capable of studying target engagement in a cellular context and provide a link between drug and cellular phenotype. Mass spectrometry based strategies provide an excellent way to study drug-target interactions as well as drug effects in a cellular context with excellent sensitivity and depth. In order to perform unbiased drug target screening several methods have been developed over the last years. In this review, we discuss affinity pull-down approaches to study direct drug-target interaction, methods which use alterations in protein stability as a measure for drug binding and the biological relevance of PTM enrichments to study the effect of inhibitors on cellular signalling

Histidine phosphorylation in human cells; a needle or phantom in the haystack?

It has been suggested that in mammalian cells histidine residues in proteins may become as frequently phosphorylated as serine, threonine and tyrosine, and may play a key role in mammalian signaling. Here we applied a robust workflow that earlier allowed us to detect histidine phosphorylation in bacteria unambiguously, to probe for histidine phosphorylation in four human cell lines. Initially, seemingly hundreds of protein histidine phosphorylations were picked up in all studied human cell lines. However, careful examination of the data, and several control experiments, led us to the conclusion that >99% of these initially assigned pHis sites were not genuine, and should be site localized to neighboring Ser/Thr residues. Nevertheless, our methods are selective enough to detect just a handful of genuine pHis sites in mammalian cells, representing well-known enzymatic intermediates. Consequently, we do not find any evidence in our data supporting that protein histidine phosphorylation plays a role in mammalian signaling

Thermal Proteome Profiling in Zebrafish Reveals Effects of Napabucasin on Retinoic Acid Metabolism

Thermal proteome profiling (TPP) allows for the unbiased detection of drug-target protein engagements in vivo. Traditionally, 1 cell type is used for TPP studies, with the risk of missing important differentially expressed target proteins. The use of whole organisms would circumvent this problem. Zebrafish embryos are amenable to such an approach. Here, we used TPP on whole zebrafish embryo lysate to identify protein targets of napabucasin, a compound that may affect signal transducer and activator of transcription 3 (Stat3) signaling through an ill-understood mechanism. In zebrafish embryos, napabucasin induced developmental defects consistent with inhibition of Stat3 signaling. TPP profiling showed no distinct shift in Stat3 upon napabucasin treatment, but effects were detected on the oxidoreductase, Pora, which might explain effects on Stat3 signaling. Interestingly, thermal stability of several aldehyde dehydrogenases was affected. Moreover, napabucasin activated aldehyde dehydrogenase enzymatic activity in vitro. Aldehyde dehydrogenases have crucial roles in retinoic acid metabolism, and functionally, we validated napabucasin-mediated activation of the retinoic acid pathway in zebrafish in vivo. We conclude that TPP profiling in whole zebrafish embryo lysate is feasible and facilitates direct correlation of in vivo effects of small molecule drugs with their protein targets

Oxygen-Releasing Biomaterials: Current Challenges and Future Applications

Oxygen is essential for the survival, function, and fate of mammalian cells. Oxygen tension controls cellular behaviour via metabolic programming, which in turn controls tissue regeneration, stem cell differentiation, drug metabolism, and numerous pathologies. Thus, oxygen-releasing biomaterials represent a novel and unique strategy to gain control over a variety of in vivo processes. Consequently, numerous oxygen-generating or carrying materials have been developed in recent years, which offer innovative solutions in the field of drug efficiency, regenerative medicine, and engineered living systems. In this review, we discuss the latest trends, highlight current challenges and solutions, and provide a future perspective on the field of oxygen-releasing materials

Steering Stem Cell Fate within 3D Living Composite Tissues Using Stimuli-Responsive Cell-Adhesive Micromaterials

Engineered living microtissues such as cellular spheroids and organoids have enormous potential for the study and regeneration of tissues and organs. Microtissues are typically engineered via self-assembly of adherent cells into cellular spheroids, which are characterized by little to no cell–material interactions. Consequently, 3D microtissue models currently lack structural biomechanical and biochemical control over their internal microenvironment resulting in suboptimal functional performance such as limited stem cell differentiation potential. Here, this work report on stimuli-responsive cell-adhesive micromaterials (SCMs) that can self-assemble with cells into 3D living composite microtissues through integrin binding, even under serum-free conditions. It is demonstrated that SCMs homogeneously distribute within engineered microtissues and act as biomechanically and biochemically tunable designer materials that can alter the composite tissue microenvironment on demand. Specifically, cell behavior is controlled based on the size, stiffness, number ratio, and biofunctionalization of SCMs in a temporal manner via orthogonal secondary crosslinking strategies. Photo-based mechanical tuning of SCMs reveals early onset stiffness-controlled lineage commitment of differentiating stem cell spheroids. In contrast to conventional encapsulation of stem cell spheroids within bulk hydrogel, incorporating cell-sized SCMs within stem cell spheroids uniquely provides biomechanical cues throughout the composite microtissues’ volume, which is demonstrated to be essential for osteogenic differentiation