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Bromodomains (BRDs) are conserved protein interaction modules which recognize (read) acetyl-lysine modifications, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan and demonstrate the utility of the set identifying roles of BRDs in cellular processes and potential translational applications. For instance, we discovered crosstalk between histone acetylation and the glycolytic pathway resulting in a vulnerability of breast cancer cell lines under conditions of glucose deprivation or GLUT1 inhibition to inhibition of BRPF2/3 BRDs. This chemical probe-set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.
Fusion of mitochondrial outer membranes is crucial for proper organelle function and involves large GTPases called mitofusins. The discrete steps that allow mitochondria to attach to one another and merge their outer membranes are unknown. By combining an in vitro mitochondrial fusion assay with electron cryo-tomography (cryo-ET), we visualize the junction between attached mitochondria isolated from Saccharomyces cerevisiae and observe complexes that mediate this attachment. We find that cycles of GTP hydrolysis induce progressive formation of a docking ring structure around extended areas of contact. Further GTP hydrolysis triggers local outer membrane fusion at the periphery of the contact region. These findings unravel key features of mitofusin-dependent fusion of outer membranes and constitute an important advance in our understanding of how mitochondria connect and merge.
The integrated stress response (ISR) is a central cellular adaptive program that is activated by diverse stressors including ER stress, hypoxia and nutrient deprivation to orchestrate responses via activating transcription factor 4 (ATF4). We hypothesized that ATF4 is essential for the adaptation of human glioblastoma (GB) cells to the conditions of the tumor microenvironment and is contributing to therapy resistance against chemotherapy. ATF4 induction in GB cells was modulated pharmacologically and genetically and investigated in the context of temozolomide treatment as well as glucose and oxygen deprivation. The relevance of the ISR was analyzed by cell death and metabolic measurements under conditions to approximate aspects of the GB microenvironment. ATF4 protein levels were induced by temozolomide treatment. In line, ATF4 gene suppressed GB cells (ATF4sh) displayed increased cell death and decreased survival after temozolomide treatment. Similar results were observed after treatment with the ISR inhibitor ISRIB. ATF4sh and ISRIB treated GB cells were sensitized to hypoxia-induced cell death. Our experimental study provides evidence for an important role of ATF4 for the adaptation of human GB cells to conditions of the tumor microenvironment characterized by low oxygen and nutrient availability and for the development of temozolomide resistance. Inhibiting the ISR in GB cells could therefore be a promising therapeutic approach.
Cells respond to protein misfolding and aggregation in the cytosol by adjusting gene transcription and a number of post-transcriptional processes. In parallel to functional reactions, cellular structure changes as well; however, the mechanisms underlying the early adaptation of cellular compartments to cytosolic protein misfolding are less clear. Here we show that the mammalian ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP), if freed from chaperones during acute stress, can dock on cellular membranes thus performing a proteostasis sensor function. We reconstituted this process in vitro and found that mainly phosphatidic acid and phosphatidylinositol-4-phosphate enhance association of chaperone-free CHIP with liposomes. HSP70 and membranes compete for mutually exclusive binding to the tetratricopeptide repeat domain of CHIP. At new cellular locations, access to compartment-specific substrates would enable CHIP to participate in the reorganization of the respective organelles, as exemplified by the fragmentation of the Golgi apparatus (effector function).
Protein disulfide isomerases (PDIs) support endoplasmic reticulum redox protein folding and cell-surface thiol-redox control of thrombosis and vascular remodeling. The family prototype PDIA1 regulates NADPH oxidase signaling and cytoskeleton organization, however the related underlying mechanisms are unclear. Here we show that genes encoding human PDIA1 and its two paralogs PDIA8 and PDIA2 are each flanked by genes encoding Rho guanine-dissociation inhibitors (GDI), known regulators of RhoGTPases/cytoskeleton. Evolutionary histories of these three microsyntenic regions reveal their emergence by two successive duplication events of a primordial gene pair in the last common vertebrate ancestor. The arrangement, however, is substantially older, detectable in echinoderms, nematodes, and cnidarians. Thus, PDI/RhoGDI pairing in the same transcription orientation emerged early in animal evolution and has been largely maintained. PDI/RhoGDI pairs are embedded into conserved genomic regions displaying common cis-regulatory elements. Analysis of gene expression datasets supports evidence for PDI/RhoGDI coexpression in developmental/inflammatory contexts. PDIA1/RhoGDIα were co-induced in endothelial cells upon CRISP-R-promoted transcription activation of each pair component, and also in mouse arterial intima during flow-induced remodeling. We provide evidence for physical interaction between both proteins. These data support strong functional links between PDI and RhoGDI families, which likely maintained PDI/RhoGDI microsynteny along > 800-million years of evolution.
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.
Heterogeneous regulation of bacterial natural product biosynthesis via a novel transcription factor
(2016)
Biological diversity arises among genetically equal subpopulations in the same environment, a phenomenon called phenotypic heterogeneity. The life cycle of the enteric bacterium Photorhabdus luminescens involves a symbiotic interaction with nematodes as well as a pathogenic association with insect larvae. P. luminescens exists in two distinct phenotypic forms designated as primary (1°) and secondary (2°). In contrast to 1° cells, 2° cells are non-pigmented due to the absence of natural compounds, especially anthraquinones (AQs). We identified a novel type of transcriptional regulator, AntJ, which activates expression of the antA-I operon responsible for AQ production. AntJ heterogeneously activates the AQ production in single P. luminescens 1° cells, and blocks AQ production in 2° cells. AntJ contains a proposed ligand-binding WYL-domain, which is widespread among bacteria. AntJ is one of the rare examples of regulators that mediate heterogeneous gene expression by altering activity rather than copy number in single cells.
Chromosomal translocations (CTs) are a genetic hallmark of cancer. They could be identified as recurrent genetic aberrations in hemato-malignancies and solid tumors. More than 40% of all “cancer genes” were identified in recurrent CTs. Most of these CTs result in the production of oncofusion proteins of which many have been studied over the past decades. They influence signaling pathways and/or alter gene expression. However, a precise mechanism for how these CTs arise and occur in a nearly identical fashion in individuals remains to be elucidated. Here, we performed experiments that explain the onset of CTs: (1) proximity of genes able to produce prematurely terminated transcripts, which lead to the production of (2) trans-spliced fusion RNAs, and finally, the induction of (3) DNA double-strand breaks which are subsequently repaired via EJ repair pathways. Under these conditions, balanced chromosomal translocations could be specifically induced. The implications of these findings will be discussed.
Hematopoietic stem cell transplantation (HSCT) is the therapeutic concept to cure the blood/immune system of patients suffering from malignancies, immunodeficiencies, red blood cell disorders, and inherited bone marrow failure syndromes. Yet, allogeneic HSCT bear considerable risks for the patient such as non-engraftment, or graft-versus host disease. Transplanting gene modified autologous HSCs is a promising approach not only for inherited blood/immune cell diseases, but also for the acquired immunodeficiency syndrome. However, there is emerging evidence for substantial heterogeneity of HSCs in situ as well as ex vivo that is also observed after HSCT. Thus, HSC gene modification concepts are suggested to consider that different blood disorders affect specific hematopoietic cell types. We will discuss the relevance of HSC heterogeneity for the development and manufacture of gene therapies and in exemplary diseases with a specific emphasis on the key target HSC types myeloid-biased, lymphoid-biased, and balanced HSCs.
Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.