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Leukemia patients bearing the t(4;11)(q21;q23) translocations can be divided into two subgroups: those expressing both reciprocal fusion genes, and those that have only the MLL-AF4 fusion gene. Moreover, a recent study has demonstrated that patients expressing both fusion genes have a better outcome than patients that are expressing the MLL-AF4 fusion protein alone. All this may point to a clonal process where the reciprocal fusion gene AF4-MLL could be lost during disease progression, as this loss may select for a more aggressive type of leukemia. Therefore, we were interested in unraveling the decisive role of the AF4-MLL fusion protein at an early timepoint of disease development. We designed an experimental model system where the MLL-AF4 fusion protein was constitutively expressed, while an inducible AF4-MLL fusion gene was induced for only 48 h. Subsequently, we investigated genome-wide changes by RNA- and ATAC-Seq experiments at distinct timepoints. These analyses revealed that the expression of AF4-MLL for only 48 h was sufficient to significantly change the genomic landscape (transcription and chromatin) even on a longer time scale. Thus, we have to conclude that the AF4-MLL fusion protein works through a hit-and-run mechanism, probably necessary to set up pre-leukemic conditions, but being dispensable for later disease progression.
Leukemia patients bearing t(6;11)(q27;q23) translocations can be divided in two subgroups: those with breakpoints in the major breakpoint cluster region of MLL (introns 9–10; associated mainly with AML M1/4/5), and others with breakpoints in the minor breakpoint cluster region (introns 21–23), associated with T-ALL. We cloned all four of the resulting fusion genes (MLL-AF6, AF6-MLL, exMLL-AF6, AF6-shMLL) and subsequently transfected them to generate stable cell culture models. Their molecular function was tested by inducing gene expression for 48 h in a Doxycycline-dependent fashion. Here, we present our results upon differential gene expression (DGE) that were obtained by the “Massive Analyses of cDNA Ends” (MACE-Seq) technology, an established 3′-end based RNA-Seq method. Our results indicate that the PHD/BD domain, present in the AF6-MLL and the exMLL-AF6 fusion protein, is responsible for chromatin activation in a genome-wide fashion. This led to strong deregulation of transcriptional processes involving protein-coding genes, pseudogenes, non-annotated genes, and RNA genes, e.g., LincRNAs and microRNAs, respectively. While cooperation between the MLL-AF6 and AF6-MLL fusion proteins appears to be required for the above-mentioned effects, exMLL-AF6 is able to cause similar effects on its own. The exMLL-AF6/AF6-shMLL co-expressing cell line displayed the induction of a myeloid-specific and a T-cell specific gene signature, which may explain the T-ALL disease phenotype observed in patients with such breakpoints. This again demonstrated that MLL fusion proteins are instructive and allow to study their pathomolecular mechanisms.
Leukemia patients bearing t(6;11)(q27;q23) translocations can be divided in two subgroups: those with breakpoints in the major breakpoint cluster region of MLL (introns 9–10; associated mainly with AML M1/4/5), and others with breakpoints in the minor breakpoint cluster region (introns 21–23), associated with T-ALL. We cloned all four of the resulting fusion genes (MLL-AF6, AF6-MLL, exMLL-AF6, AF6-shMLL) and subsequently transfected them to generate stable cell culture models. Their molecular function was tested by inducing gene expression for 48 h in a Doxycycline-dependent fashion. Here, we present our results upon differential gene expression (DGE) that were obtained by the “Massive Analyses of cDNA Ends” (MACE-Seq) technology, an established 3′-end based RNA-Seq method. Our results indicate that the PHD/BD domain, present in the AF6-MLL and the exMLL-AF6 fusion protein, is responsible for chromatin activation in a genome-wide fashion. This led to strong deregulation of transcriptional processes involving protein-coding genes, pseudogenes, non-annotated genes, and RNA genes, e.g., LincRNAs and microRNAs, respectively. While cooperation between the MLL-AF6 and AF6-MLL fusion proteins appears to be required for the above-mentioned effects, exMLL-AF6 is able to cause similar effects on its own. The exMLL-AF6/AF6-shMLL co-expressing cell line displayed the induction of a myeloid-specific and a T-cell specific gene signature, which may explain the T-ALL disease phenotype observed in patients with such breakpoints. This again demonstrated that MLL fusion proteins are instructive and allow to study their pathomolecular mechanisms.
The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death
(2023)
Every day, living organisms are challenged by internal and external factors that threaten to bring imbalance to their tightly regulated systems and disrupt homeostasis, leading to degeneration, and ultimately death. More than ever, we face the challenge of combating diseases such as COVID-19 caused by infection with the SARS-CoV-2 coronavirus. It is therefore crucial to identify host factors that control antiviral defense mechanisms. In addition, in the fight against cancer, it is becoming increasingly important to identify markers that could be used for targeted therapy to influence cellular processes and determine cell fate.
As a deubiquitylating enzyme, ubiquitin specific peptidase 22 (USP22) mediates the removal of the small molecule ubiquitin, which is post-translationally added to target proteins, thereby regulating several important processes such as protein degradation, activation or localization. Through its deubiquitylating function, USP22 controls several biological processes such as cell cycle regulation, proliferation and cancer immunoresistance by modulating key proteins involved in these pathways. Lately, USP22 was reported to positively regulate TNFα-mediated necroptosis, an inflammatory type of programmed cell death, in various human tumor cell lines by affecting RIPK3 phosphorylation. In addition, USP22 as a part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcription complex is known to regulate gene expression by removing ubiquitin from histones H2A and H2B. However, little is known about the role of USP22 in global gene expression.
In this study, we performed a genome-wide screen in the human colon carcinoma cell line HT-29 and identified USP22 as a key negative regulator of basal interferon (IFN) expression. We further demonstrated that the absence of USP22 results in increased STING activity and ubiquitylation, both basally and in response to stimulation with the STING agonist 2'3'-cGAMP, thereby affecting IFNλ1 expression and basal expression of antiviral ISGs. In addition, we were able to establish USP22 as a critical host factor in controlling SARS-CoV-2 infection by regulating infection, replication, and the generation of infectious virus particles, which we attribute in part to its role in regulating STING signaling.
In the second part of the study, we connected the findings of USP22-dependent regulation of IFN signaling and TNFα-induced necroptosis and investigated the role of USP22 during necroptosis induced by the synergistic action of IFN and the Smac mimetic BV6 in caspase-deficient settings. We identified USP22 as a negative regulator of IFN-induced necroptosis, which does not depend on STING expression, but relies on a yet unknown mechanism.
In summary, we identify USP22 as an important regulator of IFN signaling with important implications for the defense against viral infections and regulation of the necroptotic pathway that could be exploited for devising targeted therapeutic strategies against viral infections and related diseases like COVID-19, and advancing precision medicine in cancer treatment.
The stress-dependent dynamics of Saccharomyces cerevisiae tRNA and rRNA modification profiles
(2021)
RNAs are key players in the cell, and to fulfil their functions, they are enzymatically modified. These modifications have been found to be dynamic and dependent on internal and external factors, such as stress. In this study we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) to address the question of which mechanisms allow the dynamic adaptation of RNA modifications during stress in the model organism S. cerevisiae. We found that both tRNA and rRNA transcription is stalled in yeast exposed to stressors such as H2O2, NaAsO2 or methyl methanesulfonate (MMS). From the absence of new transcripts, we concluded that most RNA modification profile changes observed to date are linked to changes happening on the pre-existing RNAs. We confirmed these changes, and we followed the fate of the pre-existing tRNAs and rRNAs during stress recovery. For MMS, we found previously described damage products in tRNA, and in addition, we found evidence for direct base methylation damage of 2′O-ribose methylated nucleosides in rRNA. While we found no evidence for increased RNA degradation after MMS exposure, we observed rapid loss of all methylation damages in all studied RNAs. With NAIL-MS we further established the modification speed in new tRNA and 18S and 25S rRNA from unstressed S. cerevisiae. During stress exposure, the placement of modifications was delayed overall. Only the tRNA modifications 1-methyladenosine and pseudouridine were incorporated as fast in stressed cells as in control cells. Similarly, 2′-O-methyladenosine in both 18S and 25S rRNA was unaffected by the stressor, but all other rRNA modifications were incorporated after a delay. In summary, we present mechanistic insights into stress-dependent RNA modification profiling in S. cerevisiae tRNA and rRNA.
Currently, due to the misuse of antibiotics, we are facing a major public health problem. The resistance to antibiotics of certain bacterial strains makes the treatment of infections very complex.
In this context, the present thesis project concerns the study of a bacterial efflux complex capable of transporting antibiotics from the cytoplasm to the outside of the cell. This complex is composed of an inner-membrane Major Facilitator Superfamily (MFS) transporter (EmrB, E. coli multidrug resistance), a channel of the outer membrane TolC (Tolerance to Colicin E1) and a periplasmic adapter (EmrA, E. coli multidrug resistance). Unlike RND-type efflux systems (such as AcrAB-TolC), little is known about the MFS-type EmrAB-TolC system. It is therefore important to study the entire complex on a structural and functional level, to analyse the marked differences between these two types of transport systems. The goal of my thesis project was to study at least one EmrAB-TolC complex from a structural point of view. For my studies the aim was to isolate the complex directly from bacteria overexpressing the three protein partners. In a first step, 15 homologous EmrAB-TolC systems were identified and their corresponding genes amplified from genomic DNA of different Gram-negative bacteria. Among the genes of the 15 systems, the genes coding for the E. coli and V. cholerae systems were further studied. The expression vectors encoded fluorescent markers for the monitoring of the expression levels of different proteins and for studying the formation of complexes. In a first step, the different protein expression levels (EmrB-mRFP1 and EmrA-sfGFP) were studied for several expression strains of E. coli by measuring the red and green fluorescence levels and by Western blot (anti-His, Myc, and Strep for EmrB, EmrA, and TolC). The E. coli strain C41(DE3) was best suited for co-expression of EmrAB-TolC. In a second step, the FSEC (Fluorescence detection Size Exclusion Chromatography) methodology was used to identify a complex suitable for structural study. Thus this method enabled the observation that the EmrAB-TolC complex of E. coli was produced in higher amount than that of V. cholerae. The final co-purification protocol consists in perfoming a gentle lysis of the bacteria using lysozyme, then after solubilization with DDM, the purification is started by a Ni2+-NTA affinity chromatography step followed by a size exclusion chromatography step. Finally, the fractions containing the three protein partners are used for the detergent-exchange by amphipol A8-35 before the structural study by electron microscopy. Negative stain EM-micrographs displayed elongated objects with a length of 33 nm in side view. An average image of EmrAB-TolC shows similarities to that of the AcrAB-TolC complex observed under similar conditions. Similarities included the characteristic densities of TolC. Whereas differences were found in the lower part of EmrAB which is thinner than the lower part of AcrAB. The densities visible above the amphipol-ring correspond to EmrA, which displays a channel-like structure as in AcrA. The channel however seems to extend further towards the amphipol belt. Since EmrB does not have an extended periplasmic domain as the RND proteins have, these densities are therefore solely assigned to EmrA. EmrA, on the other side, contacts TolC akin to the interaction of AcrA/MexA to their cognate outer membrane channels (TolC/OprM) in a ‘tip-to-tip’ fashion.
The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.
Background: Zolpidem is a non-benzodiazepine hypnotic agent which has been shown to be effective in inducing and maintaining sleep in adults and is one of the most frequently prescribed hypnotics in the world. For drugs that are used to treat sleeping disorders, the time to reach the maximum concentration (Tmax) of the drug in plasma is important to achieving a fast onset of action and this must be maintained when switching from one product to another.
Objectives: The main objective of the present work was to create a PBPK/PD model for zolpidem and establish a clinically relevant “safe space” for dissolution of zolpidem from the commercial immediate release (IR) formulation. A second objective was to analyze literature pharmacokinetic data to verify the negative food effect ascribed to zolpidem and consider its ramifications in terms of the “safe space” for dissolution.
Methods: Using dissolution, pharmacokinetic and pharmacodynamic data, an integrated PBPK/PD model for immediate release zolpidem tablets was constructed in Simcyp®. This model was used to identify the clinically relevant dissolution specifications necessary to ensure efficacy.
Results: According to the simulations, as long as 85% of the drug is released in 45 minutes or less, the impact on the PK and PD profiles of zolpidem would be minimal. According to the FDA, the drug has to dissolve from the test and reference products at a similar rate and to an extent of 85% in not more than 30 minutes to pass bioequivalence via the BCS-biowaiver test. Thus, the BCS-biowaiver specifications are somewhat more stringent than the “safe space” based on the PBPK/PD model. Published data from fasted and fed state pharmacokinetic studies suggest but do not prove a negative food effect of zolpidem.
Conclusions: A PBPK/PD model indicates that current BCS biowaiver criteria are more restrictive for immediate release zolpidem tablets than they need to be. In view of the close relationship between PK and PD, it remains advisable to avoid taking zolpidem tablets with or immediately after a meal, as indicated by the Stilnox® labeling.
The endosteal bone marrow niche and vascular endothelial cells provide sanctuaries to leukemic cells. In murine chronic myeloid leukemia (CML) CD44 on leukemia cells and E-selectin on bone marrow endothelium are essential mediators for the engraftment of leukemic stem cells (LSC). We hypothesized that non-adhesion of CML-initiating cells to E-selectin on the bone marrow endothelium may lead to superior eradication of LSC in CML after treatment with imatinib than imatinib alone. Indeed, here we show that treatment with the E-selectin inhibitor GMI-1271 in combination with imatinib prolongs survival of mice with CML via decreased contact time of leukemia cells with bone marrow endothelium. Non-adhesion of BCR-ABL1+ cells leads to an increase of cell cycle progression and an increase of expression of the hematopoietic transcription factor and protooncogene Scl/Tal1 in leukemia-initiating cells (LIC). We implicate SCL/TAL1 as indirect phosphorylation target of BCR-ABL1 and as a negative transcriptional regulator of CD44 expression. We show that increased SCL/TAL1 expression is associated with improved outcome in human CML. These data demonstrate the BCR-ABL1-specific, cell-intrinsic pathways leading to altered interactions with the vascular niche via the modulation of adhesion molecules - a strategy therapeutically exploitable in future.
The new class of microbial rhodopsins, called xenorhodopsins (XeRs),[1] extends the versatility of this family by inward H+ pumps.[2–4] These pumps are an alternative optogenetic tool to the light-gated ion channels (e.g. ChR1,2), because the activation of electrically excitable cells by XeRs is independent from the surrounding physiological conditions. In this work we functionally and spectroscopically characterized XeR from Nanosalina (NsXeR).[1] The photodynamic behavior of NsXeR was investigated on the ps to s time scale elucidating the formation of the J and K and a previously unknown long-lived intermediate. The pH dependent kinetics reveal that alkalization of the surrounding medium accelerates the photocycle and the pump turnover. In patch-clamp experiments the blue-light illumination of NsXeR in the M state shows a potential-dependent vectoriality of the photocurrent transients, suggesting a variable accessibility of reprotonation of the retinal Schiff base. Insights on the kinetically independent switching mechanism could furthermore be obtained by mutational studies on the putative intracellular H+ acceptor D220.