Throughout their lives, organisms maintain homeostasis in the face of changing conditions by interpreting environmental signals and translating them into appropriate biochemical and biological outputs. Crucial for all these processes is the correct operation of signaling pathways in cells and tissues. We study how signaling mechanisms impact homeostasis on molecular, cellular and organismal level.
Highlights from our research groups
It takes two to tango: using the ubiquitin fold to turn on secretion
September 26, 2019
Protein Kinase D (PKD) is an enzyme at the heart of many cellular functions. By modifying other proteins, it controls the trafficking of essential cargo in the sorting center of the cell, the Golgi apparatus. During his PhD in Thomas Leonard’s lab, Daniel Elsner has identified a ubiquitin-like domain in PKD that plays a crucial role in its activation. The findings, published in the Journal of Biological Chemistry, revise our understanding of how the “on-switch” of PKD is wired in the cell.
At the heart of protein sorting in the cell, the Golgi apparatus is the organelle that sorts proteins and lipids into vesicles, so that they can be transported to their intra- and extracellular destinations. At the center of this process, PKD regulates the biogenesis of the vesicles used for cargo transportation. Like all protein kinases, PKD adds phosphate groups to its target proteins in a process called phosphorylation. In doing so, it can control their location, activity, or cellular functions. Because of their central role in controlling information flow in the cell, the activity of protein kinases needs to be tightly regulated. Hyperactivation or inactivation can perturb essential cellular processes.
PKD is activated by the lipid diacylglycerol (DAG), a small membrane-embedded molecule that acts as a signal that can be recognized, interpreted, and transduced into work in the cell. While recognition of DAG by PKD is relatively well understood, the mechanism by which it turns on the kinase has been in equal parts mysterious and controversial. One possibility was dimerization-mediated auto-activation, a common mechanism exploited by many eukaryotic kinases in which two copies of the kinase self-associate to modify each other. However, no biochemical or structural evidence supported such a mechanism. Alternatively, another group of protein kinases, of the protein kinase C (PKC) family, was proposed to activate PKD.
The team of Thomas Leonard has now demonstrated that a previously unannotated ubiquitin like domain (ULD) in PKD drives its dimerization. Their report is the first example of the use of a ubiquitin-like domain to mediate homo-dimerization. With this discovery, the scientists have been able to redraw the wiring diagram by which PKD is activated. In their proposed model, PKD is maintained in an inactive conformation in the cell until it is recruited to the membrane by DAG. As a consequence of local concentration, PKD dimerizes with the help of its ubiquitin-like domain. This physical association of two PKD molecules, and not other kinases, then allows PKD to autophosphorylate and thereby propagate the signal. As in the idiom “it takes two to tango”, PKD can only propagate the signal with a partner.
These findings increase the functional repertoire of ubiquitin-like domains as well as provide important mechanistic insight into the activation of PKD. They also have important implications for rationalizing disease-associated mutations, dominant negative effects of, over-expression and, perhaps most importantly, understanding vesicle biogenesis in the secretory pathway.
Original Publication in Journal of Biological Chemistry:
Daniel J. Elsner, Katharina M. Siess, Thomas Gossenreiter, Markus Hartl, Thomas A. Leonard: A ubiquitin-like domain controls Protein Kinase D dimerization and activation by trans-autophosphorylation. https://doi.org/10.1074/jbc.RA119.008713
CDK8 & CDK19 – Twin enzymes with non-twin roles in defense against viruses
September 05, 2019
The enzyme CDK8 and its paralog CDK19 are essential modules of the Mediator, a large protein complex that coordinates several key steps in transcription. CDK8 and CDK19 are highly similar and were thought to be functionally redundant. The group of Pavel Kovarik now discovered that CDK8/CDK19 are actually mechanistically distinct and activate different sets of genes in the interferon-induced anti-viral response. The results revise our understanding of anti-viral immunity and could help develop novel therapies of immune disorders. The findings are published in Molecular Cell.
Interferons are a group of signaling proteins that are at the forefront of the innate and adaptive immune systems. When an organism gets infected, they dock on receptors on the cells surface and initiate a cascade of signals that ultimately causes the cell to activate antiviral or antibacterial gene expression. Gene expression itself requires the Mediator complex, a crucial component of transcription initiation. Mediator connects RNA polymerase and gene-specific transcription factors. Like a complex machine it consists of different modules, each with a distinct role. A module containing the kinases CDK8/CDK19 is part of this machine and modifies other molecules. By doing so, it positively or negatively regulates transcription, depending on the gene. This way, the CDK8/CDK19 module precisely adjusts the transcription output to meet the needs.
CDK8 and CDK19 share up to 77% of their amino acid sequences, and have been thought to act like twins, virtually interchangeable and functionally redundant. The team of Pavel Kovarik, in close collaboration with Dylan Taatjes from the University of Colorado Boulder now first set out to identify the distinct functions of the two enzymes by analyzing their role in response to one type of interferon, called interferon gamma. Surprisingly they found out that the two enzymes are more like fraternal twins, highly similar, but functionally and mechanistically distinct. Both CDK8 and CDK19 are key regulators in the interferon gamma response, but activate different sets of genes. Also, only CDK8 acts as a kinase, while CDK19 has a scaffolding function, as first author Iris Steinparzer explains in detail: "Given the high similarity of CDK8 and CDK19, especially within their almost identical kinase domains, we were highly surprised that CDK8 and CDK19 cannot substitute for each other in launching the interferon-induced antiviral program. An even bigger surprise was the finding that CDK8 acts as enzyme (=kinase) while CDK19 does not need its enzymatic activity. We are now curious to figure out the molecular basis for these astonishing differences."
Due to their important role in transcription and also cell proliferation, chemical inhibitors of CDK8 and CDK19 are being tested in medically relevant approaches. The currently available inhibitors target both kinases indiscriminately. The discovery of distinct functions of CDK8 and CDK19 implies that targeting the enzymes separately might decisively improve effectiveness and application of CDK8/CDK19 inhibitors.
Original Publication in Molecular Cell
Iris Steinparzer, Vitaly Sedlyarov, Jonathan D. Rubin, Kevin Eislmayr, Matthew D. Galbraith, Cecilia B. Levandowski, Terezia Vcelkvoa, Lucy Sneezum, Florian Wascher, Fabian Amman, Renata Kleinova, Heather Bender, Zdenek Andrysik, Joaquin M. Espinosa, Giulio Superti-Furga, Robin D. Dowell, Dylan J. Taatjes, Pavel Kovarik: Transcriptional Responses to IFN-γ Require Mediator Kinase-Dependent Pause Release and Mechanistically Distinct CDK8 and CDK19 Functions. https://doi.org/10.1016/j.molcel.2019.07.034
How cells quickly activate innate immunity
July 02, 2019
Upon infection cells manage to quickly switch from normal operation to immune reaction in a matter of minutes. This innate immunity requires a cellular signal cascade that activates antimicrobial or antiviral gene expression. Scientists led by Thomas Decker at the Max Perutz Labs have discovered that an alternative version of the activator of antimicrobial gene expression is constantly present on DNA. A molecular switch between the alternative and the regular version enables a quick onset of the immune response. The findings are published in the journal “Nature Communications”.
Defense against pathogens is based on pre-existing and acquired mechanisms. Both innate and acquired immunity relies on signaling proteins called cytokines. A subgroup of these, called interferons dock on cell surface receptors to initiate a signaling process that causes the cell to activate genes responsible for immune defense against microbes. These signals stimulate activation of the protein complex ISGF3, responsible for initiating antimicrobial gene expression. The team of Thomas Decker now found out that two of the three proteins forming this complex are in fact constantly located at these genes, independently of the activating signals caused by interferons.
This ‘light’ version of ISGF3 maintains a low expression of antimicrobial genes, similar to an engine running in first gear. When activated by interferons, the complete version of ISGF3 complex assembles inside the nucleus, switching the gears and revving up the genetic machine of the innate immune system. The proximity of the proteins to the DNA and the rapid exchange of ‘light’ and complete versions explains how the innate immune system activates in such a quick manner, as Postdoctoral researcher and first author of the paper, Ekaterini Platanitis concludes: “When studying why antimicrobial gene expression is never completely switched off we were fascinated to find modular use of ISGF3 components as a way of switching between cellular alertness and immunological activation. This is a fascinating example of molecular economy. Cells maintain a low amount of innate immunity that does not interfere with their normal, healthy physiology. Upon infection the ISGF3 switch allows them to rapidly devote their entire genetic program to the combat against invading pathogens.”
Publication in Nature Communications:
Ekaterini Platanitis, Duygu Demiroz, Anja Schneller, Katrin Fischer, Christophe Capelle, Markus Hartl, Thomas Gossenreiter, Mathias Müller, Maria Novatchkova & Thomas Decker A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription doi.org/10.1038/s41467-019-10970-y
Renovating the house – how cells stay in good shape
March 08, 2019
Everyone owning a house knows it: to stay like new it needs cleaning and mending. Similarly cells constantly renovate and get rid of unwanted material in a process called autophagy in order to replace it with new parts. This ensures that the organism stays healthy over the years. Like a house renovation, different contractors are employed with repairs and getting rid of waste, and perfect communication is required between them. An international team of scientists from Berlin and Berkeley led by Sascha Martens from the Max Perutz Labs, a joint venture University of Vienna and the Medical University of Vienna now describe how this communication between two important factors takes place and thus ensures that autophagy correctly works in the cell.
Among others, the cell employs these two “contractors”, proteins called p62 and FIP200 to fulfil certain jobs. FIP200 is an important factor in the formation of the autophagosome, the “waste bag” where cellular trash is engulfed. The protein p62 collects and prepares the trash, so that the autophagosome can form around it. The scientists discovered that the two proteins directly communicate with each other. Impairment of this communication disturbs the whole process of autophagy. Structural analysis of FIP200 protein revealed that a part of it is shaped like a claw. Like a worker would grab a pile of trash the claw interacts with p62 and the collected cargo.
First author Eleonora Turco explains the findings in detail: "We characterized the interaction between p62 and FIP200 combining biochemistry, structural biology and cell biology techniques. We discovered that p62 not only recognizes and prepares the cargo, but, through the interaction with FIP200, is able to attract the autophagy machinery leading to the formation of an autophagosome and finally degradation of the cargo. In collaboration with Oliver Daumke, Marie Witt and Tobias Bock-Bierbaum from the Max Delbrück Center in Berlin and the group of James Hurley in Berkeley we identified a pocket in FIP200 Claw that binds a small motif of p62, revealing a long-sought link between cargo collection and autophagic degradation."
Cells are the building blocks of life, who among other functions, constantly decode the genetic information embedded in DNA and produce proteins, needed for many functions in the organism. Like humans sometimes tend to clutter their homes with unwanted or broken things, the cell produces broken or unneeded proteins that start to accumulate in it. Keeping the “cellular house” clean and new is therefore essential for the organism, as faulty autophagy is involved in many human diseases. Mutations in p62 cause a number of diseases including neurodegeneration. Therefore, the understanding of the mechanisms behind autophagy could help understand how certain diseases in humans occur.
Eleonora Turco, Marie Witt, Christine Abert, Tobias Bock-Bierbaum, Ming-Yuan Su, Riccardo Trapannone, Martin Sztacho, Alberto Danieli, Xiaoshan Shi, Gabriele Zaffagnini, Annamaria Gamper, Martina Schuschnig, Dorotea Fracchiolla, Daniel Bernklau, Julia Romanov, Markus Hartl, James H. Hurley, Oliver Daumke, Sascha Martens FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates doi.org/10.1016/j.molcel.2019.01.035
RNA helicase activity in the immune system: a matter of sex chromosomes
February 11, 2019
The proverbial differences between sexes are also found in many biological processes. Immune responses for example have for a long time been known to have distinct differences between males and females. Scientists from Thomas Decker’s group in collaboration with other research institutes now add another piece in the puzzle of sex related differences in immunity.
When human organisms are infected by pathogens, a cascade of molecular signals makes sure the immune system correctly reacts to the invaders. On a genetic level so called RNA helicases play an important role. These enzymes are capable of “unpacking” the genome by separating the strands of the DNA double helix but also catalyse metabolic processes of RNA including the synthesis of transcripts of antimicrobial genes. The scientists created organisms lacking the RNA helicase DDX3X. Their results show that DDX3X supports decoding of genes, thus making important contributions to the activation of macrophages, essential white blood cells that engulf and destroy pathogens. Also, production of certain white blood cells is impaired causing an inability to combat infection with bacteria. Thus, DDX3X plays an essential role in the antibacterial immune response.
The DDX3X gene has a counterpart on the male Y chromosome called DDX3Y. This means that male organisms lacking DDX3X still have a copy of DDX3Y. Male organisms survived a lack of DDX3X better than their female counterparts, indicating that that the presence of the Y Chromosome is able to compensate for the loss of DDX3X. The role of the male RNA helicase and its function in other bacteria remains elusive, yet the two enzymes share a high degree of similarity, hinting that DDX3Y could be a key factor in differences in innate immunity in humans.
“It has been known for many years that antibacterial immune responses differ between sexes. Known causes for this difference include immunological activities of sex hormones or different microbiota. With DDX3X we were able to identify a monogenic cause of immunological differences between sexes. Future studies must reveal in how far our results translate to humans and whether they are of relevance for gender-tailored manipulation of the immune system during diseases”, group leader Thomas Decker concludes.
Szappanos D, Tschismarov R, Perlot T, Westermayer S, Fischer K, Platanitis E, Kallinger F1, Novatchkova M, Lassnig C, Müller M, Sexl V, Bennett KL, Foong-Sobis M, Penninger JM, Decker T. The RNA helicase DDX3X is an essential mediator of innate antimicrobial immunity. DOI: 10.1371/journal.ppat.1007397
Autophagy’s little helpers: How proteins mediate autophagosome-vacoule fusion
August 13, 2018
During autophagy the cell collects, degrades and recycles unwanted cellular material. This is an important process as cellular waste is ultimately harmful to the whole organism if it accumulates in the cells. Analogous to the processing of household waste, this mechanism requires certain protagonists and elements. Scientists from group leader Claudine Kraft’s Lab at Max F. Perutz Laboratories together with colleagues from the University of Freiburg have now gained new insight into the role of proteins in autophagosome-vacuole fusion.
In autophagy, damaged cell parts, unused proteins or other cellular waste are engulfed within a vesicle called the autophagosome, just like we put our household waste in bags. These “bags” are then transported to a lysosome in mammals or the vacuole in yeast and plants. These organelles serve a similar purpose to recycling factories: they degrade the material brought by autophagosomes so that the building blocks can be reused. Just like trucks, sanitation and factory workers keeping the process of waste disposal going in our world, autophagy-related proteins initiate and regulate the mechanism on the molecular level in cells. Over 40 have been identified to this day, providing an insight into the process from the formation, up to the maturation of the autophagosome. But how do autophagosomes and vacuoles achieve to correctly fuse?
In a paper published in the Journal of Cell Biology (JCB), Claudine Kraft’s group now discovered a possible answer. To understand the requirements of autophagosome-vacuole fusion the scientists recreated the process in the lab. They separated vacuoles, autophagosomes and intracellular fluid from yeast cells and created an environment where the fusion can be observed in vitro, i.e. outside a living organism. One common mechanism in cells for membrane fusion requires bundles of four so-called SNARE proteins. The scientists verified that autophagosome-vacuole fusion is a SNARE-driven process, and confirmed that three previously known SNAREs act during the fusion event. Importantly, they also discovered the fourth SNARE required, Ykt6. First author Levent Bas explains: “Our novel in vitro approach allowed us for the first time to clearly show that the three known SNAREs act on the vacuolar membrane during autophagosome-vacuole fusion. It also enabled us to identify the elusive fourth SNARE, Ykt6, and to show that it acts on the autophagosomal side.”
The findings help further the understanding of autophagy and its underlying molecular processes. But the employed in vitro approach that took the scientists two years to develop and refine holds a lot of potential for future research: “The method will help unravel the mechanisms of autophagosome-vacuole fusion in greater detail and, of course, identify additional proteins acting in the fusion process”, Levent Bas concludes.
Publication in JCB
Levent Bas, Daniel Papinski, Mariya Licheva, Raffaela Torggler, Sabrina Rohringer, Martina Schuschnig, Claudine Kraft: Reconstitution reveals Ykt6 as the autophagosomal SNARE in autophagosome–vacuole fusion.
A system of check and balances in the blood
May 24, 2018
Hematopoietic Stem Cells (HSCs) give rise to blood and immune cells of the body, and are therefore essential for our survival. They are in a dormant state, but whenever new blood needs to be formed, such as after blood loss or chemotherapy, they are rapidly activated to compensate for the loss. After completing their mission, they need to go back to their dormant state. The group of Manuela Baccarini at the Max F. Perutz Laboratories, a joint venture of the University of Vienna and the Medical University of Vienna, has now shown how intracellular signalling can safeguard this delicate balance between activation and dormancy. Their results are published in the prominent journal Cell Stem Cell.
Blood is the juice of life, as while circulating through the body it delivers vital substances such as oxygen and nutrients to cells and tissues.Chemotherapy, radiotherapy and blood loss in general impoverish the system. A special kind of cells in the bone marrow, called hematopoietic stem cells (HSCs), is able to replenish the impoverished system by giving rise not only to red blood cells, but also to cells of the immune system. Thus, HSCs play an absolutely crucial role for survival. To compensate for blood loss, HSCs, which are usually dormant, start to actively self-renew and differentiate into all blood cell types. After completing their task, however, HSCs need to revert back to their dormant state very rapidly, or they will exhaust. This requires a very delicate balance. A small tilt towards activation or dormancy can have catastrophic consequences for the organism, resulting, in the worst case, in death.
Manuela Baccarini’s group at the Max Perutz Labs has now discovered the mechanism behind this delicate balance. First author Christian Baumgartner clarifies: “Up until now, we knew that the balance between activation and return to dormancy existed and was essential, but not how it was kept and which players were involved”.The new paper identifies the players and details their regulation during stress-induced blood production. “Two pivotal intracellular signalling pathways, almost always activated in parallel, are coordinated by a feedback loop that keeps HSCs in perfect balance. The beauty of it is that the system will be reset irrespectively of the stimulus that initiated it,” says Manuela Baccarini. To prove this, the group deliberately removed one of the players from the feedback loop and found that the entire balance was shifted, resulting in unrestrained HSC activation, exhaustion of the HSC compartment, and ultimately in the failure to produce enough blood cells to compensate for the loss.
Inhibitors of the pathway at study are currently being used in cancer therapy. The work of the Baccarini now shows that these compounds could be repurposed to mobilize “lazy” HSCs, as seen for instance in ageing organisms.
Publication in Cell Stem Cell
Christian Baumgartner, Stefanie Toifl, Matthias Farlik, Florian Halbritter, Ruth Scheicher, Irmgard Fischer, Veronika Sexl, Christoph Bock, and Manuela Baccarini: An ERK-Dependent Feedback Mechanism prevents Hematopoietic Stem Cell exhaustion. Cell Stem Cell. DOI: 10.1016/j.stem.2018.05.003 https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(18)30221-2.
Immune system can be modulated by targeted manipulation of cell metabolism
August 21, 2017
In its attempt to fight a serious bacterial infection, caused by listeria, for example, the immune system can become so over-activated that the resulting inflammatory response and its consequences can quickly lead to death. Scientists from the Medical University of Vienna and the Max F. Perutz Laboratories of the MedUni Vienna and the University of Vienna, supervised by Gerhard Zlabinger from the Institute of Immunology, have now demonstrated in an animal model that such an excessive response by the immune system can be modulated by targeted manipulation of the sugar metabolism to produce an immune response that efficiently eliminates the pathogens without causing any harmful secondary reactions.
This is achieved by using a specific sugar compound during the infection, so-called 2-Deoxy-D-glucose (2-DG). This sugar molecule differs from glucose in that it lacks a hydroxyl group, consisting of one hydrogen atom and one oxygen atom. Administering 2-DG, which serves to inhibit glycolysis, causes increased production of interleukin-12 (IL-12), a pro-inflammatory cytokine, while suppressing the production of interleukin-10, an anti-inflammatory cytokine.
One of the main functions of IL-12 is to trigger a specific T-cell immune response, which is part of the cellular immune response, to specifically boost the defence mechanisms that serve to eliminate intracellular pathogens (such as listeria, for example). "The administration of 2-DG apparently changes the nature of the immune response and hence also the intensity of the inflammatory process," explains Gerhard Zlabinger, MedUni Vienna immunologist. "Inflammation is modulated so that the immune system is once again able to manage the situation itself, without allowing the infection to have a lethal outcome. It is as if we are pressing the reset button, thereby preventing excessive inflammatory responses."
The underlying mechanism is not yet known. According to Johannes Kovarik, lead author of the study, the expression of IL-12 , which can be formed by two separate chains (p35 and/or p40), probably plays a role. Following administration of 2-DG, only p40 is upregulated, while no p35 is produced. This modified cytokine profile (there is also a simultaneous increase in IL-23 expression) could subsequently bring about modified recruitment of immune cells and hence have a decisive influence upon the intensity of the immune response.
"Our study is an excellent example of the importance of the immune metabolism, that is to say the modified metabolism of essential nutrients by activated immune system cells," explains Max Perutz Labs Group Leader Thomas Decker. "We were amazed by the extent to which mice became resistant to bacterial infections due to inhibition of the glucose metabolism. This metabolic reversal creates a push-pull effect: increased production of activating mediators with simultaneous inhibition of immunosuppressive mediators."
Manipulation of the sugar metabolism and associated modulation of the immune system could open up new options for treating specific infectious and also autoimmune diseases in the future. Further research work will, of course, be required for this – some projects are already being started at MedUni Vienna's Institute of Immunology.
Publication in PLOS ONE:
"Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis."
J.J. Kovarik, E. Kernbauer, M.A. Hölzl, J. Hofer, G.A. Gualdoni, K.G. Schmetterer, F. Miftari, Y. Sobanov, A. Meshcheryakova, D. Mechtcheriakova, N. Witzeneder, G. Greiner, A. Ohradanova-Repic, P. Waidhofer-Söllner, M.D. Säemann, T. Decker, G.J. Zlabinger. doi.org/10.1371/journal.pone.0180900.