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Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.
Zinc (Zn2+) can modulate platelet and coagulation activation pathways, including fibrin formation. Here, we studied the (patho)physiological consequences of abnormal platelet Zn2+ storage and release. To visualize Zn2+ storage in human and mouse platelets, the Zn2+ specific fluorescent dye FluoZin3 was used. In resting platelets, the dye transiently accumulated into distinct cytosolic puncta, which were lost upon platelet activation. Platelets isolated from Unc13d−/− mice, characterized by combined defects of α/δ granular release, showed a markedly impaired Zn2+ release upon activation. Platelets from Nbeal2−/− mice mimicking Gray platelet syndrome (GPS), characterized by primarily loss of the α-granule content, had strongly reduced Zn2+ levels, which was also confirmed in primary megakaryocytes. In human platelets isolated from patients with GPS, Hermansky-Pudlak Syndrome (HPS) and Storage Pool Disease (SPD) altered Zn2+ homeostasis was detected. In turbidity and flow based assays, platelet-dependent fibrin formation was impaired in both Nbeal2−/− and Unc13d−/− mice, and the impairment could be partially restored by extracellular Zn2+. Altogether, we conclude that the release of ionic Zn2+ store from secretory granules upon platelet activation contributes to the procoagulant role of Zn2+ in platelet-dependent fibrin formation.
The NEDD8-activating enzyme (NAE) inhibitor MLN4924 inhibits cullin-RING ubiquitin ligase complexes including the SKP1-cullin-F-box E3 ligase βTrCP. MLN4924 therefore inhibits also the βTrCP-dependent activation of the classical and the alternative NFĸB pathway. In this work, we found that a subgroup of multiple myeloma cell lines (e.g., RPMI-8226, MM.1S, KMS-12BM) and about half of the primary myeloma samples tested are sensitized to TNF-induced cell death by MLN4924. This correlated with MLN4924-mediated inhibition of TNF-induced activation of the classical NFκB pathway and reduced the efficacy of TNF-induced TNFR1 signaling complex formation. Interestingly, binding studies revealed a straightforward correlation between cell surface TNFR1 expression in multiple myeloma cell lines and their sensitivity for MLN4924/TNF-induced cell death. The cell surface expression levels of TNFR1 in the investigated MM cell lines largely correlated with TNFR1 mRNA expression. This suggests that the variable levels of cell surface expression of TNFR1 in myeloma cell lines are decisive for TNF/MLN4924 sensitivity. Indeed, introduction of TNFR1 into TNFR1-negative TNF/MLN4924-resistant KMS-11BM cells, was sufficient to sensitize this cell line for TNF/MLN4924-induced cell death. Thus, MLN4924 might be especially effective in myeloma patients with TNFR1+ myeloma cells and a TNFhigh tumor microenvironment.
Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a thus far unknown cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the lytic-latent switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily drugable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.
The N,C-coupled naphthylisoquinoline alkaloid ancistrocladinium A belongs to a novel class of natural products with potent antiprotozoal activity. Its effects on tumor cells, however, have not yet been explored. We demonstrate the antitumor activity of ancistrocladinium A in multiple myeloma (MM), a yet incurable blood cancer that represents a model disease for adaptation to proteotoxic stress. Viability assays showed a potent apoptosis-inducing effect of ancistrocladinium A in MM cell lines, including those with proteasome inhibitor (PI) resistance, and in primary MM cells, but not in non-malignant blood cells. Concomitant treatment with the PI carfilzomib or the histone deacetylase inhibitor panobinostat strongly enhanced the ancistrocladinium A-induced apoptosis. Mass spectrometry with biotinylated ancistrocladinium A revealed significant enrichment of RNA-splicing-associated proteins. Affected RNA-splicing-associated pathways included genes involved in proteotoxic stress response, such as PSMB5-associated genes and the heat shock proteins HSP90 and HSP70. Furthermore, we found strong induction of ATF4 and the ATM/H2AX pathway, both of which are critically involved in the integrated cellular response following proteotoxic and oxidative stress. Taken together, our data indicate that ancistrocladinium A targets cellular stress regulation in MM and improves the therapeutic response to PIs or overcomes PI resistance, and thus may represent a promising potential therapeutic agent.
Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1A-33G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non-glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor’s glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera.
Introduction
Pro-thrombotic events are one of the prevalent causes of intensive care unit (ICU) admissions among COVID-19 patients, although the signaling events in the stimulated platelets are still unclear.
Methods
We conducted a comparative analysis of platelet transcriptome data from healthy donors, ICU, and non-ICU COVID-19 patients to elucidate these mechanisms. To surpass previous analyses, we constructed models of involved networks and control cascades by integrating a global human signaling network with transcriptome data. We investigated the control of platelet hyperactivation and the specific proteins involved.
Results
Our study revealed that control of the platelet network in ICU patients is significantly higher than in non-ICU patients. Non-ICU patients require control over fewer proteins for managing platelet hyperactivity compared to ICU patients. Identification of indispensable proteins highlighted key subnetworks, that are targetable for system control in COVID-19-related platelet hyperactivity. We scrutinized FDA-approved drugs targeting indispensable proteins and identified fostamatinib as a potent candidate for preventing thrombosis in COVID-19 patients.
Discussion
Our findings shed light on how SARS-CoV-2 efficiently affects host platelets by targeting indispensable and critical proteins involved in the control of platelet activity. We evaluated several drugs for specific control of platelet hyperactivity in ICU patients suffering from platelet hyperactivation. The focus of our approach is repurposing existing drugs for optimal control over the signaling network responsible for platelet hyperactivity in COVID-19 patients. Our study offers specific pharmacological recommendations, with drug prioritization tailored to the distinct network states observed in each patient condition. Interactive networks and detailed results can be accessed at https://fostamatinib.bioinfo-wuerz.eu/.
Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.
DNA alkyltransferase and alkyltransferase-like family proteins are responsible for the repair of highly mutagenic and cytotoxic O\(^6\)-alkylguanine and O\(^4\)-alkylthymine bases in DNA. Their mechanism involves binding to the damaged DNA and flipping the base out of the DNA helix into the active site pocket in the protein. Alkyltransferases then directly and irreversibly transfer the alkyl group from the base to the active site cysteine residue. In contrast, alkyltransferase-like proteins recruit nucleotide excision repair components for O\(^6\)-alkylguanine elimination. One or more of these proteins are found in all kingdoms of life, and where this has been determined, their overall DNA repair mechanism is strictly conserved between organisms. Nevertheless, between species, subtle as well as more extensive differences that affect target lesion preferences and/or introduce additional protein functions have evolved. Examining these differences and their functional consequences is intricately entwined with understanding the details of their DNA repair mechanism(s) and their biological roles. In this review, we will present and discuss various aspects of the current status of knowledge on this intriguing protein family.
The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; Braf\(^{CA}\); Pten\(^{lox/+}\) melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but they were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.