Podcasts about ptms

In this episode, podcast co-hosts Dr. Dwight Stoll and Dr. James Grinias talk with Dr. Benjamin Garcia. Dr. Garcia is the Raymond H. Wittcoff Distinguished Professor and Head of the Department of Biochemistry and Molecular Biophysics in the School of Medicine at Washington University in St. Louis. His research interests primarily focus on the development of mass spectrometry-based proteomic approaches for the analysis of post-translational modifications of proteins (PTMs) and modified proteomes. His group specializes in high-throughput and quantitative analysis of many classes of modified proteins, especially epigenetic histone PTMs. He is a prolific author and scientific leader, with an h-index approaching 120. He has received a LOT of awards to date, but we sat down with him at the Eastern Analytical Symposium where he was recognized for his recent EAS Award for Outstanding Achievement in Mass Spectrometry. In the conversation we talk about the origins of Dr. Garcia's interests in analytical science, mass spectrometry, and biomedical research, and he shares his thoughts about the current state and near future for technologies used for biomolecule separations including peptides, proteins, and oligonucleotides. We also discuss Dr. Garcia's highly impactful efforts to promote participation in the analytical sciences by people from all backgrounds, and he shares some thoughts about the “to-do list” for his upcoming term as chair of the ACS Division of Analytical Chemistry.

BUFFALO, NY- August 27, 2024 – A new #research perspective was #published in Aging (listed by MEDLINE/PubMed as "Aging (Albany NY)" and "Aging-US" by Web of Science), Volume 16, Issue 15 on July 25, 2024, entitled, “Trioxidized cysteine and aging: a molecular binomial that extends far beyond classical proteinopathic paradigms.” Oxidative stress (OS) - characterized by an imbalance between oxidants and antioxidants - leads to the formation of oxidative posttranslational modifications (PTMs), including those involving cysteine (Cys) residues in aging proteomes. Specifically, the formation of trioxidized Cys (t-Cys) results in permanent protein damage. Recent findings in rodents have revealed that irregular regulation of t-Cys residues in the aging proteome disrupts homeostatic phosphorylation signaling, leading to alterations in proteins similar to those caused by phosphorylated serine (p-Ser) residues. In this perspective, researchers José Antonio Sánchez Milán, María Mulet, Aida Serra and Xavier Gallart-Palau from University Hospital Arnau de Vilanova (HUAV) and University of Lleida (UdL), present novel data, validating the increase of specific t-Cys sites associated with aging in a blood-related circulating human proteome. "The scope and findings included here support the hypothesis that t-Cys residues may serve as important mechanistic and biological markers, warranting further exploration in the context of unhealthy aging and age-related major diseases.” DOI - https://doi.org/10.18632/aging.206036 Corresponding authors - Aida Serra - aida.serra@udl.cat, and Xavier Gallart-Palau - xgallart@irblleida.cat Video short - https://www.youtube.com/watch?v=roO_8WMGak8 Sign up for free Altmetric alerts about this article - https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.206036 Subscribe for free publication alerts from Aging - https://www.aging-us.com/subscribe-to-toc-alerts Keywords - aging, oxidative stress, unhealthy aging, t-Cys, aging diseases, aging proteome About Aging-US The mission of the journal is to understand the mechanisms surrounding aging and age-related diseases, including cancer as the main cause of death in the modern aged population. The journal aims to promote 1) treatment of age-related diseases by slowing down aging, 2) validation of anti-aging drugs by treating age-related diseases, and 3) prevention of cancer by inhibiting aging. (Cancer and COVID-19 are age-related diseases.) Please visit our website at https://www.Aging-US.com​​ and connect with us: Facebook - https://www.facebook.com/AgingUS/ X - https://twitter.com/AgingJrnl Instagram - https://www.instagram.com/agingjrnl/ YouTube - https://www.youtube.com/@AgingJournal LinkedIn - https://www.linkedin.com/company/aging/ Pinterest - https://www.pinterest.com/AgingUS/ Spotify - https://open.spotify.com/show/1X4HQQgegjReaf6Mozn6Mc MEDIA@IMPACTJOURNALS.COM

Episode Summary: In episode 185 of the Aerospace Advantage, Tomorrow's F-35: Cooling is Key, host Heather “Lucky” Penney and Doug Birkey chat with Matt Pess, Chief Engineer on the Enhanced Power and Cooling System (EPACS) program at Collins Aerospace, about a key technology to sustain the F-35's future capability growth. The fighter's powerful sensor suite, computing power, and avionics set it apart as a hugely capable twenty-first century combat aircraft. Operating these technologies demands robust cooling via a key aircraft subsystem known as power thermal management system (PTMS), especially as new systems are fielded on subsequent versions of the aircraft. The Joint Program Office (JPO) recently launched an effort to explore a new generation of PTMS systems. EPACS is one of the options. Big picture, this is a good news story because it means the F-35 is radically expanding what it can deliver in the battlespace. Realizing these gains will demand innovation. We discuss these factors with a key expert. This episode will explore this evolution and how the Collins team is focused on a potential solution.  Credits: Host: Heather “Lucky” Penney, Senior Fellow, The Mitchell Institute for Aerospace Studies Producer: Shane Thin Executive Producer: Douglas Birkey Guest: Douglas Birkey, Executive Director, The Mitchell Institute for Aerospace Studies Guest: Matt Pess, Chief Engineer, Enhanced Power and Cooling System (EPACS) Program, Collins Aerospace Related Reading: Accelerating 5th Generation Airpower: Bringing Capability and Capacity to the Merge Links: Subscribe to our Youtube Channel: https://bit.ly/3GbA5Of Website: https://mitchellaerospacepower.org/ Twitter: https://twitter.com/MitchellStudies Facebook: https://www.facebook.com/Mitchell.Institute.Aerospace LinkedIn: https://bit.ly/3nzBisb Instagram: https://www.instagram.com/mitchellstudies/ #MitchellStudies #AerospaceAdvantage #Allies #Sweden #Europe Thank you for your continued support!

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550331v1?rss=1 Authors: Yucel, B. P., Al Momany, E., Evans, A. J., Seager, R., Wilkinson, K., Henley, J. M. Abstract: Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550533v1?rss=1 Authors: Ceglowski, J., Hoffman, H. K., Hoff, K. J., McCurdy, B. L., Moore, J., Prekeris, R. Abstract: The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as, axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550360v1?rss=1 Authors: del Pino, R. C., Zoltner, M., Yamada, K., Butterfield, E. R., Field, M. Abstract: Post-translational modifications (PTMs) modulate protein function, with ubiquitylation a pre-eminent example with major roles in protein turnover. Ubiquitylation utilises a ligase enzyme cascade for conjugation of ubiquitin to client proteins and cullin-RING ligases are amongst the most complex known. We reconstructed evolution of cullin-RING E3 ubiquitin ligases across eukaryotes and experimentally characterised two cullin complexes in trypanosomatids, a taxon highly divergent from animals and fungi. We find considerable diversity within cullins and, in particular, trypanosomatids share only a minority of cullins with other lineages. Furthermore, we identify expansions in cullin client adaptor protein families, novel client adaptors and demonstrate client specificity. Finally we show that ornithine decarboxylase (TbODC), an important target of the drug trypanosome eflornithine, is a substrate for TbCul-A and overturn earlier models for eflornithine specificity. These studies highlight lineage-specific roles for cullin E3s and their contributions towards eukaryotic complexity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Prepare to be electrified by the cutting-edge world of military aviation, where the F-35 Lightning faces a unique stumbling block: its engine. We're cracking open the complexities of the Pratt Whitney F-135's cooling struggle, and how this challenge could spark an engine competition between industry titans. Strap in for a heady exploration of jet engines, their role in cooling fighter jets, and the F-35's pressing need for enhanced systems.We're not stopping at jet engines, though. You'll also get a glimpse into the world of block upgrades for the F-35, the potential game-changing ECU and PTMS upgrades from Pratt Whitney, and an innovative cooling system from Collin's Aerospace. We'll also navigate the unchartered territories of the Adaptive Engine Transition Program (AETP) and the conundrum of maintaining two different engine designs. Warning: things might get a little heated as we dive into the future of the F-35 and the rapidly evolving landscape of military aviation.To help support this podcast and become a PilotPhotog ProCast member: https://www.buzzsprout.com/1555784/supportIf you enjoy this episode, subscribe to this podcast, you can find links to most podcast streaming services here: PilotPhotog Podcast (buzzsprout.com)Sign up for the free weekly newsletter Hangar Flyingwith Tog here: https://hangarflyingwithtog.com You can check out my YouTube channel for many videos on fighter planes here:https://youtube.com/c/PilotPhotog If you'd like to support this podcast via Patreon:https://www.patreon.com/PilotPhotog And finally, you can follow me on Twitter here:https://twitter.com/pilotphotogSupport the show

A new review paper was published in Aging (Aging-US) Volume 15, Issue 8, entitled, “How can we modulate aging through nutrition and physical exercise? An epigenetic approach.” The World Health Organization predicts that by 2050, 2.1 billion people worldwide will be over 60 years old, a drastic increase from only 1 billion in 2019. Considering these numbers, strategies to ensure an extended “healthspan” or healthy longevity are urgently needed. In this new review, researchers Ana Teresa Rajado, Nádia Silva, Filipa Esteves, David Brito, Alexandra Binnie, Inês M. Araújo, Clévio Nóbrega, José Bragança, and Pedro Castelo-Branco from the ALFA Score Consortium, University of Algarve Campus Gambelas, William Osler Health System, and Champalimaud Centre for the Unknown discuss their present study that approaches the promotion of healthspan from an epigenetic perspective. Epigenetic phenomena are modifiable in response to an individual's environmental exposures, and therefore link an individual's environment to their gene expression pattern. Epigenetic studies demonstrate that aging is associated with decondensation of the chromatin, leading to an altered heterochromatin structure, which promotes the accumulation of errors. “In this article we explore aging and its associated epigenetic changes as well as how these changes may be delayed or reversed through nutrition, caloric restriction and sustained physical activity, as schematized in Figure 2.” Canonical histones are replaced by histone variants, concomitant with an increase in histone post-translational modifications (PTMs). A slight increase in DNA methylation at promoters has been observed, which represses transcription of previously active genes, in parallel with global genome hypomethylation. Aging is also associated with deregulation of gene expression - usually provided by non-coding RNAs - leading to both the repression of previously transcribed genes and to the transcription of previously repressed genes. “Age-associated epigenetic events are less common in individuals with a healthy lifestyle, including balanced nutrition, caloric restriction and physical exercise. Healthy aging is associated with more tightly condensed chromatin, fewer PTMs and greater regulation by ncRNAs.” DOI: https://doi.org/10.18632/aging.204668 Corresponding Author: Pedro Castelo-Branco - pjbranco@ualg.pt Sign up for free Altmetric alerts about this article: https://aging.altmetric.com/details/email_updates?id=10.18632%2Faging.204666 Subscribe for free publication alerts from Aging: https://www.aging-us.com/subscribe-to-toc-alerts Keywords: epigenetics, aging, nutrition, caloric restriction, physical exercise About Aging-US Launched in 2009, Aging-US publishes papers of general interest and biological significance in all fields of aging research and age-related diseases, including cancer—and now, with a special focus on COVID-19 vulnerability as an age-dependent syndrome. Topics in Aging-US go beyond traditional gerontology, including, but not limited to, cellular and molecular biology, human age-related diseases, pathology in model organisms, signal transduction pathways (e.g., p53, sirtuins, and PI-3K/AKT/mTOR, among others), and approaches to modulating these signaling pathways. Please visit our website at https://www.Aging-US.com​​ and connect with us: SoundCloud - https://soundcloud.com/Aging-Us Facebook - https://www.facebook.com/AgingUS/ Twitter - https://twitter.com/AgingJrnl Instagram - https://www.instagram.com/agingjrnl/ YouTube - https://www.youtube.com/@AgingJournal LinkedIn - https://www.linkedin.com/company/aging/ Pinterest - https://www.pinterest.com/AgingUS/ Media Contact 18009220957 MEDIA@IMPACTJOURNALS.COM

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536711v1?rss=1 Authors: Ellis, M. J., Lekka, C., Tulmin, H., O'Brien, D. P., Dhayal, S., Zeissler, M.-L., Knudsen, J. G., Kessler, B. M., Morgan, N. G., Todd, J. A., Richardson, S. J., Stefana, I. M. Abstract: Background: The microtubule-associated protein Tau has attracted diverse and increasing research interest, with Tau being mentioned in the title/abstract of nearly 34,000 PubMed-indexed publications to date. To accelerate studies into Tau biology, the characterisation of its multiple proteoforms, including disease-relevant post-translational modifications (PTMs), and its role in neurodegeneration, a multitude of Tau-targeting antibodies have been developed, with hundreds of distinct antibody clones currently available for purchase. Nonetheless, concerns over antibody specificity and limited understanding of the performance of many of these reagents has hindered research. Methods: We have employed a range of techniques in combination with samples of murine and human origin to characterise the performance and specificity of 53 commercially-available Tau antibodies by Western blot, and a subset of these, 35 antibodies, in immunohistochemistry. Results: Continued expression of residual protein was found in presumptive Tau "knockout" human cells and further confirmed through mass-spectrometry proteomics, providing evidence of Tau isoforms generated by exon skipping. Importantly, many total and isoform-specific antibodies failed to detect this residual Tau, as well as Tau expressed at low, endogenous levels, thus highlighting the importance of antibody choice. Our data further reveal that the binding of several "total" Tau antibodies, which are assumed to detect Tau independently of post-translational modifications, was partially inhibited by phosphorylation. Many antibodies also displayed non-specific cross-reactivity, with some total and phospho-Tau antibodies cross-reacting with MAP2 isoforms, while the "oligomer-specific" T22 antibody detected monomeric Tau on Western blot. Regardless of their specificity, with one exception, the phospho-Tau antibodies tested were found to not detect the unphosphorylated protein. Conclusions: We identify Tau antibodies across all categories (total, PTM-dependent and isoform-specific) that can be employed in Western blot and/or immunohistochemistry applications to reliably detect even low levels of Tau expression with high specificity. This is of particular importance for studying Tau in non-neuronal cells and peripheral tissues, as well as for the confident validation of knockout cells and/or animal models. This work represents an extensive resource that serves as a point of reference for future studies. Our findings may also aid in the re-interpretation of existing data and improve reproducibility of Tau research. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.24.534149v1?rss=1 Authors: Donzelli, S., OSullivan, S., Mahul-Mellier, A.-L., Ulusoy, A., Fusco, G., Kumar, S. T., Chiki, A., Burtscher, J., Boussouf, M. L. D., Rostami, I., De Simone, A. D. S., Di Monte, D. A., Lashuel, H. A. Abstract: Increasing evidence points to post-translational modifications (PTMs) as key regulators of alpha-synuclein (-Syn) function in health and disease. However, whether these PTMs occur before or after -Syn pathology formation and their role in regulating -Syn toxicity remain unclear. In this study, we demonstrate that post-fibrillization nitration of -Syn fibrils induced their fragmentation, modified their surface and dynamic properties but not their structure, and nearly abolished their seeding activity in primary neurons and in vivo. Furthermore, we show that the dynamic and surface properties of the fibrils, rather than simply their length, are important determinants of -Syn fibril seeding activity. Altogether, our work demonstrates that post-aggregation modifications of -Syn may provide novel approaches to target a central process that contributes to pathology formation and disease progression. Finally, our results suggest that the pattern of PTMs on pathological aggregates, rather than simply their presence, could be a key determinant of their toxicity and neurodegeneration. This calls for reconsidering current approaches relying solely on quantifying and correlating the level of pathology to assess the efficacy of novel therapies, as not all -Syn aggregates in the brain are pathogenic. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.14.528480v1?rss=1 Authors: Vanagas, L., Munoz, D., Cristaldi, C., Ganuza, A., Najera, R., Bonardi, M. C., Turowski, V. R., Guzman, F., Deng, B., Kim, K., Sullivan, W. J., Angel, S. O. Abstract: Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by reader proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N-terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.01.518806v1?rss=1 Authors: Andreu-Carbo, M., Egoldt, C., Velluz, M.-C., Aumeier, C. Abstract: The properties of single microtubules within the microtubule network can be modulated through posttranslational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could either enter through the microtubule ends or at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen depends on kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase TAT1 is independent of kinesin-1 and shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de-)acetylation and scales with the size of the cells. The control of shaft damage represents a novel mechanism to regulate PTM inside the microtubule by giving access to the lumen. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.16.516716v1?rss=1 Authors: Cheron, J., Beccari, L., Hague, P., Icick, R., Defrance, M., Carusone, T., Despontin, C., Bhogaraju, S., Vorspan, F., Bonnefont, J., de Kerchove d'Exaerde, A. Abstract: The individual risk of developing drug addiction is highly determined by the epigenetic landscape. Chromatin remodeling regulates drug-induced transcriptional and behavioral effects and the consequent development of addictive behaviors. Several chromatin modifications in the ventral tegmental area and nucleus accumbens, including histone H3 methylation, H3 and H4 acetylation, have been implicated in drug addiction. Still, the contribution of other histones and their post-translational modifications (PTMs), such as monoubiquitination is unknown. Here we found that H2A monoubiquitination in the paraventricular thalamus (PVT) plays a major role in cocaine-adaptive behaviors and local cocaine-evoked transcriptional repression. Mice undergoing chronic cocaine administration showed a specific increased monoubiquitination of H2A. Furthermore, we showed that this histone PTM is controlled, in the PVT, by an interaction between melanoma-associated antigen D1 (Maged1), a scaffold protein involved in drug addiction, and USP7, a deubiquitinase. Accordingly, Maged1 specific inactivation in thalamic vGluT2 neurons, or USP7 inhibition, blocked cocaine-evoked H2A monoubiquitination and abolished cocaine locomotor sensitization. Finally, we identified genetic variations of MAGED1 and USP7 associated with modified transition to cocaine addiction and cocaine-induced aggressive behavior in human subjects. These findings identified a new epigenetic modification in a noncanonical reward pathway of the brain and a potent marker of epigenetic risk factor for drug addiction in humans. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.11.516192v1?rss=1 Authors: Orsini, F., Argyrousi, E. K., Restelli, E., Ford, L. K., Takamura, H., Matsuzaki, S., Zentilin, L., Pascente, R., Kanaan, N. M., Soni, R., Katayama, T., Chiesa, R., Forloni, G., Kosik, K. S., Kandel, E. R., Fraser, P. E., Arancio, O., Fioriti, L. Abstract: Abnormal intracellular accumulation of Tau aggregates is a hallmark of Alzheimer's disease (AD) and other Tauopathies, such as Frontotemporal dementia (FTD), which can be caused by mutations of Tau. Mutated and pathological Tau can undergo a range of post-translational modifications (PTMs) that might trigger or modulate disease pathology. Recent studies indicate that modification of wild type Tau by Small ubiquitin-like modifier SUMO isoform 1 (SUMO1) controls Tau hyperphosphorylation and aggregation, suggesting that SUMOylation acts as a central regulator of Tau's biochemical properties. Besides SUMO1, Tau is modified by SUMO2/3, however the consequences of this modification have not been investigated. Here, using viral approaches on primary hippocampal neurons, transgenic mice expressing mutant Tau and SUMO2, and iPSC-derived neurons from FTD patients, we evaluated whether SUMO2/3 conjugation modifies the neurodegenerative disease pathology associated with the aggregation-prone mutant Tau P301L, P301S, and R406W variants. We found that mutant forms of Tau are targets of SUMO2/3, and SUMO2/3 conjugation is neuroprotective. Importantly, expression of mutant Tau is accompanied by a significant reduction of SUMO2/3 conjugation levels, and restoring levels of SUMO2 reduces mutant Tau aggregation and phosphorylation in all model systems Furthermore, overexpression of SUMO2 restores levels of pre- and post-synaptic markers, associated with a complete rescue of the LTP and memory deficits in transgenic mice expressing mutant Tau. These findings bring to light the potential therapeutic implication of manipulating SUMO conjugation to detoxify Tau through PTM-based approaches. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512471v1?rss=1 Authors: Godinho, S. A., Monteiro, P., Yeon, B., Wallis, S. S. Abstract: Intracellular organelle organisation is conserved in eukaryotic cells and is primarily achieved through active transport by motor proteins along the microtubule cytoskeleton. Microtubule posttranslational modifications (PTMs) contribute to microtubule diversity and differentially regulate motor-mediated transport. Here we show that centrosome amplification induces a global change in organelle positioning towards the cell periphery and facilitates nuclear migration through confined spaces. This reorganisation requires kinesin-1 and is analogous to loss of dynein. Cells with amplified centrosomes display increased levels of acetylated tubulin, a PTM known to enhance kinesin-1 mediated transport. Depletion of -tubulin acetyltransferase 1 (TAT1) to block tubulin acetylation, which has no impact on control cells, rescues the displacement of centrosomes, mitochondria and vimentin, but not Golgi or endosomes. Analyses of the distribution of acetylated microtubules indicates that the polarisation of modified microtubules, rather than levels alone, plays an important role in organelle positioning. We propose that tubulin acetylation differentially impacts kinesin-1-mediated organelle displacement, suggesting that each organelle must have its own sensing and response mechanisms to ensure fine-tuning of its distribution in cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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Extremely large pre-trained language models (PTMs) such as GPT-3 are usually released as a service, allowing users to design taskspecific prompts to query the PTMs through some black-box APIs. In such a scenario, which we call Language-Model-as-a-Service (LMaaS), gradients of the PTMs are usually not available. Can we optimize the task prompts by only accessing the model inference APIs? Based on recent observations that large PTMs have a very low intrinsic dimensionality, this work proposes the Black-Box Tuning to optimize PTMs through derivativefree algorithms. 2022: Tianxiang Sun, Yunfan Shao, Hong Qian, Xuanjing Huang, Xipeng Qiu https://arxiv.org/pdf/2201.03514v1.pdf

For more details, visit #DrGPCR​​​ Podcast Episode #56 page: http://www.drgpcr.com/episode-56-with-dr-adriano-marchese/ ------------------------------------------- Adriano Marchese is a Professor of Biochemistry at the Medical College of Wisconsin. Adriano received his Bachelor of Science degree in Pharmacology in 1991 from the University of Toronto. He continued his graduate studies at the University of Toronto where he earned his MSc (1994) and Ph.D. (1998) in Pharmacology. He then moved to Thomas Jefferson University in Philadelphia, PA, for his postdoctoral training in Jeff Benovic's laboratory studying the regulation of G protein-coupled receptor trafficking and signaling. In 2004 Adriano joined the faculty of the Department of Pharmacology at Loyola University Chicago. In 2016 he decided to move his lab to the Medical College of Wisconsin in Milwaukee, WI. Adriano's research has contributed to our understanding of the role that ubiquitin plays in GPCR signaling and trafficking. His laboratory is interested in understanding the mechanisms that govern spatial and temporal regulation of GPCR signaling by -arrestins and post-translational modifications (PTMs), such as phosphorylation, ubiquitination, and SUMOylation. His lab has shown a role for -arrestins and PTMs in GPCR trafficking and signaling and has leveraged this knowledge to reveal the spatial and temporal requirements for GPCR activation of signaling pathways related to cell survival, proliferation, and migration. The ultimate goal of Adriano's research is to target novel aspects of GPCR signaling for therapeutic development. ------------------------------------------- Imagine a world in which the vast majority of us are healthy. The #DrGPCR Ecosystem is all about dynamic interactions between us who are working towards exploiting the druggability of #GPCR's. We aspire to provide opportunities to connect, share, form trusting partnerships, grow, and thrive together. To build our #GPCR Ecosystem, we created various enabling outlets. For more details, visit our website http://www.DrGPCR.com/Ecosystem/ ------------------------------------------- Are you a #GPCR professional? - Register to become a Virtual Cafe speaker http://www.drgpcr.com/virtual-cafe/ - Subscribe to our Monthly Newsletter http://www.drgpcr.com/newsletter/ - Listen and subscribe to #DrGPCR Podcasts http://www.drgpcr.com/podcast/ - Support #DrGPCR Ecosystem with your Donation. http://www.drgpcr.com/sponsors/ - Reserve your spots for the next #DrGPCR Virtual Cafe http://www.drgpcr.com/virtual-cafe/

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.379768v1?rss=1 Authors: Daly, L. A., Brownridge, P. J., See, V., Eyers, C. E. Abstract: Adaption of cells to low oxygen environments is an essential process mediated in part by the Hypoxia Inducible Factors (HIFs). Like other transcription factors, the stability and transcriptional activity of HIFs, and consequently the hypoxic response, are regulated by post-translational modification (PTM) and changes in biomolecular interactions. However, our current understanding of PTM-mediated regulation of HIFs is primarily based on in vitro protein fragment-based studies, with validation typically having been conducted by in cellulo fragment expression and hypoxia mimicking drugs. Consequently, we still lack an understanding of true oxygen deprivation signaling via HIF. Using an immunoprecipitation-based, mass spectrometry approach, we characterize the regulation of in cellulo expressed full-length HIF-1 and HIF-2, in terms of both PTM and binding partners, in response to normoxia (21% oxygen) and hypoxia (1% oxygen). These studies revealed that a change in oxygen tension significantly alters the complexity and composition of HIF- protein interaction networks, with HIF-2 in particular having an extended hypoxia-induced interactome, most notably with mitochondrial-associated proteins. Both HIF isoforms are heavily covalently modified: we define ~40 different sites of PTM on each of HIF-1 and HIF-2, comprising 13 different PTM types, including multiple cysteine modifications and a highly unusual phosphocysteine. Over 80% of the PTMs identified are novel, and approximately half exhibit oxygen-dependency under these conditions. Combined with domain and evolutionary analysis of >225 vertebrate species, we validate Ser31 phosphorylation on HIF-1 as a regulator of transcription, and propose functional roles for Thr406, Thr528 and Ser581 on HIF-2. Copy rights belong to original authors. Visit the link for more info

In this episode of the Epigenetics Podcast, we caught up with Geneviève Almouzni, Ph.D., Research Director at the CNRS at Institut Curie in Paris, to talk about her work on the regulation of chromatin organization by histone chaperones. Geneviève Almouzni got her Ph.D. from Université Pierre-et-Marie-Curie in 1988 under the supervision of Marcel Méchali. She then moved to the United States to work as a postdoc in the National Institutes of Health in the laboratory of Professor Alan Wolffe. In 1994, she returned to Paris and became a Junior Group Leader at Institut Curie and became a Group Leader there in 2000. In 2013, she took over the direction of research at the Institut Curie and became the third woman to hold this position, after Marie Curie and Irène Joliot-Curie. Geneviève Almouzni’s research focuses on the assembly of chromatin and the identification of histone chaperones. Histone chaperones are necessary for the establishment and maintenance of chromatin, as they help to assemble the nucleosomes out of the core histones and DNA. This occurs both when the polymerase transcribes through a nucleosome and after DNA replication and repair. The Almouzni group has identified and characterized multiple histone chaperones, including CAF-1, HirA, and HJURP. Furthermore, they investigated how post-translational modifications on soluble histones influence the final epigenetic state of the nucleosome and the reassembly of chromatin after DNA replication. In the last couple of years, the group has focused on the unraveling the link between the structure of chromatin at centromeres and cancer. In this interview, we discuss the focus of the Almouzni lab on histone chaperones, how the lab was able to identify its first one with CAF-1, how histone PTMs on soluble histones influence the deposition on the DNA, and how the chromatin on centromeres is involved in cancer.   References   Dominique Ray-Gallet, Jean-Pierre Quivy, … Geneviève Almouzni (2002) HIRA Is Critical for a Nucleosome Assembly Pathway Independent of DNA Synthesis (Molecular Cell) DOI: 10.1016/S1097-2765(02)00526-9 Pierre-Henri L. Gaillard, Emmanuelle M.-D. Martini, … Geneviève Almouzni (1996) Chromatin Assembly Coupled to DNA Repair: A New Role for Chromatin Assembly Factor I (Cell) DOI: 10.1016/S0092-8674(00)80164-6 Jean-Pierre Quivy, Danièle Roche, … Geneviève Almouzni (2004) A CAF-1 dependent pool of HP1 during heterochromatin duplication (The EMBO Journal) DOI: 10.1038/sj.emboj.7600362   Contact   Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on Linked-In Active Motif on Facebook eMail: podcast@activemotif.com  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.13.295089v1?rss=1 Authors: Ueda, H. Abstract: Advances in Nanopore single-molecule direct RNA sequencing (DRS) have presented the possibility of detecting comprehensive post-transcriptional modifications (PTMs) as an alternative to experimental approaches combined with high-throughput sequencing. It has been shown that the DRS method can detect the change in the raw electric current signal of a PTM; however, the accuracy and reliability still require improvement. Here, we presented a new software, called nanoDoc, for detecting PTMs from DRS data using a deep neural network. Current signal deviations caused by PTMs are analyzed via Deep One-Class Classification with a convolutional neural network. Using a ribosomal RNA dataset, the software archive displayed an area under the curve (AUC) accuracy of 0.96 for the detection of 23 different kinds of modifications in Escherichia coli and Saccharomyces cerevisiae. We also demonstrated a tentative classification of PTMs using unsupervised clustering. Finally, we applied this software to severe acute respiratory syndrome coronavirus 2 data and identified commonly modified sites among three groups. nanoDoc is open source (GPLv3) and available at https://github.com/uedaLabR/nanoDoc . Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.31.270165v1?rss=1 Authors: Zhu, Z., Han, Z., Halabelian, L., Yang, X., Ding, J., Zhang, N., Ngo, L., Song, J., Zeng, H., He, M., Zhao, Y., Arrowsmith, C. H., Luo, M., Bartlett, M. G., Zheng, Y. G. Abstract: Short-chain acylation of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity, especially p300 and HAT1. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.26.269043v1?rss=1 Authors: Supekar, N. T., Shajahan, A., Gleinich, A., Rouhani, D., Heiss, C., Azadi, P. Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID 19, a vaccine to protect against deadly COVID 19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O glycosylated and also contains significant levels of N glycosylation. We were able to confirm the presence of O glycans on seven sites with substantial glycan occupancy, in addition to less abundant O glycans on four sites. We also detected N glycans on two out of five potential N glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.21.261818v1?rss=1 Authors: Meyer, J. G. Abstract: Shotgun proteomics techniques infer the presence and quantity of proteins using peptide proxies, which are produced by cleavage of all isolated protein by a protease. Most protein quantitation strategies assume that multiple peptides derived from a protein will behave quantitatively similar across treatment groups, but this assumption may be false for biological or technical reasons. Here, I describe a strategy called peptide correlation analysis (PeCorA) that detects quantitative disagreements between peptides mapped to the same protein. Simple linear models are used to assess whether the slope of a peptide's change across treatment groups differs from the slope of all other peptides assigned to the same protein. Reanalysis of proteomic data from primary mouse microglia with PeCorA revealed that about 15% of proteins contain one discordant peptide. Inspection of the discordant peptides shows utility of PeCorA for direct and indirect detection of regulated PTMs, and also for discovery of poorly quantified peptides that should be excluded. PeCorA can be applied to an arbitrary list of quantified peptides, and is freely available as a script written in R. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.23.217968v1?rss=1 Authors: Chiki, A., Ricci, J., Hegde, R. N., Abriata, L. A., Reif, A., Lashuel, H. A. Abstract: Posttranslational modifications (PTMs) within the first 17 amino acids (Nt17) of exon1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntingtons disease (HD). In this manuscript, we describe a new and simple methodology for producing milligram quantities of highly pure wild type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK); and, 2) the development of an efficient methodology for producing recombinant native Httex1 proteins using a SUMO-fusion expression and purification strategy. As proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site- specifically phosphorylated and fluorescently labeled or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 structure, aggregation, interactome and function(s) in health and disease. Copy rights belong to original authors. Visit the link for more info

In this episode of the CUNA News podcast, Crystal Long and Diana Dykstra, winners of the National Credit Union Foundation’s Herb Wegner Memorial Award for Individual Achievement, share their feelings on winning the award and the paths they followed to earn them such a distinguished honor.Crystal Long is president/CEO of GECU in El Paso, Texas. Under her leadership, GECU has made a commitment to community development and enabled many El Paso residents achieve economic prosperity.She is also a dedicated advocate for the credit union movement who puts her staff and members first. Long started her career with GECU as a file clerk at age 19.Among her accomplishments, Long spearheaded the award-winning GECU Neighborhood Branch™ expansion strategy to serve members via personal teller machines (PTMs) and on-site loan officers in areas where financial services options are limited. Not only are the branches in more convenient locations for members to access, the PTMs allow for extended hours of operation.GECU financial coaches also provide free and personalized counseling, financial education seminars, and financial literacy training to thousands of members each year. And it has launched initiatives such as the Volunteer Income Tax Assistance (VITA) Program, which helps local families file their taxes for free and keep their entire refund.Diana Dykstra is president/CEO of the California and Nevada Credit Union Leagues. Throughout her career, Diana has dedicated herself to creating innovative partnerships that change both the competitive landscape and the lives of others.Dykstra has been involved in several pioneering projects that continue to reverberate throughout the credit union industry. As senior vice president at Golden 1 Credit Union, Dykstra led her team to develop the prototype of the CU Direct Lending program in 1992. For more than 20, CU Direct has been a lending technology leader, helping credit unions fund $211 billion in loans.In 2014, Dykstra was instrumental in launching the California and Nevada Credit Union Leagues Financial Capability Initiative—powered by EverFi—which provides innovative online financial literacy programs to drive large-scale change at credit unions and the increase financial capability of their members.The Foundation also honored Nusenda Credit Union Foundation with an Outstanding Organization Award for its innovation and cooperative leadership in creating their Co-op Capital Program, uplifting the Albuquerque community.And it recognized Brett Martinez, president/CEO of Redwood Credit Union and CUNA chair, with an Anchor Award for his exemplary leadership during the 2017 California Wildfires.

Eukaryotic genomes are organized inside the cell nucleus in a structured macromolecular DNA-protein polymer named chromatin, formed by single discrete unites called Nucleosomes. The packing of the genetic information into chromatin allows the efficient regulation of several nuclear processes, such as gene expression and transcription, DNA replication, cell cycle progression, chromosome segregation and DNA damage repair. Chromatin comes in two flavors: a transcriptionally active, more loosened state, called euchromatin and a transcriptionally silent or low expressed, more compact state, called heterochromatin. The assembly of silent chromatin or heterochromatin is fundamental for the regulation of every nuclear process and it is driven in most Eukaryotes by the deposition and the read-out of the histone H3 lysine 9 methylation (H3K9me) post-translational modification (PTM). H3K9me on the nucleosome is specifically bound by chromatin readers called chromodomains (CD) and this recognition is fundamental for the downstream processes that lead to the formation of heterochromatin and shut down the expression of single genes or entire gene clusters. Despite several studies have been done on different chromodomains binding to H3K9me histone tail peptides, to date there was no structural information on how chromodomains interact with their natural binding partners, the H3K9me3 Nucleosomes. In a preliminary structural study carried out in our laboratory we solved the cryo-electron microscopy (Cryo-EM) structure of the chromodomain of the fission yeast Chp1 protein (Chp1CD) in complex with an H3K9me nucleosome. The structure showed that the Chp1CD interacts not only with the histone H3 tail but also with the histone globular domains in the Nucleosome core, primarily with histone H3. Mutations in the residues of Chp1CD that form the binding interface with the Nucleosome core (two loops in the β-sheet of the domain) caused a drop of the affinity in vitro for the H3K9me Nucleosome, which was independent from the histone H3K9me tail interaction. Cells harboring the same Chp1CD loop mutations were defective in silencing centromeric transcripts and maintain the deposition of the H3K9me mark for heterochromatin formation. This indicated that Chp1CD-nucleosome core interaction is fundamental for heterochromatin formation in fission yeast and opened up to the possibility that chromodomains could read multiple histone PTMs, on both the recruiting histone tail and on the nucleosome core. This study substantially contributes to understand how chromodomains interact with chromatin, how much the nucleosome core interaction is conserved among different CDs and how different chromodomain proteins are regulated at the same loci. Understanding how chromodomain readers recognize nucleosomes is fundamental to uncover the basics of gene silencing and heterochromatin formation.

In the mammalian genome, cytosine methylation (5mC) plays a central role in the epigenetic regulation of gene expression and has been implicated in a variety of biological processes, including genome stability, imprinting or differentiation. Compared to other epigenetic marks, DNA methylation has been thought to be relatively stable. However, genome-wide loss of 5mC, or DNA demethylation, has been observed in specific developmental stages and in various types of cancer. The discovery of the TET family of enzymes in 2009 was a watershed moment in comprehending the mechanisms of DNA demethylation. TET proteins oxidize 5mC to 5- hydroxymethylcytosine (5hmC), 5-formlycytosine (5fC) and 5-carboxylcytosine (5caC), which not only serve as key intermediates in active DNA demethylation pathways, but can also act as independent epigenetic marks. In this study, various aspects of TET-mediated DNA demethylation have been intensively investigated. Using quantitative mass-spectrometry-based proteomics readers for the different cytosine derivatives in mouse embryonic stem cells (ESCs), neuronal progenitor cells, and adult mouse brain tissue were identified. Readers for these modifications are only partially overlapping and are dynamic during differentiation. Moreover, the oxidized derivatives of 5mC recruit distinct transcription regulators as well as a large number of DNA repair proteins, implicating DNA damage response as the main pathway contributing to active DNA demethylation. To identify additional non-canonical DNA bases, highly sensitive quantitative mass-spectrometry led to the discovery of 5-hydroxymethyluracil (5hmU) in ESCs. Genomic 5hmU is not generated via deamination of 5hmC, as widely suggested, but through direct oxidation of thymine by TET proteins. In addition, screening for specific 5hmU readers identified different transcriptional and epigenetic factors, implicating that this mark has a specific function in ESCs. So far, only little is known how TET enzymes are regulated and how they are modified by posttranslational modifications (PTMs). Mapping TET phosphorylation and glycosylation sites at amino acid resolution revealed that these PTMs are interdependent and mostly occur at regulatory protein regions. Finally, a reporter gene based assay could demonstrate that in vitro methylation causes gene silencing while subsequent oxidation, resulting in DNA demethylation, leads to gene reactivation in vivo. Different knockout and rescue experiments clearly show that oxidation of methylcytosine by TET proteins and subsequent removal by TDG or NEIL glycosylases and the base excision repair pathway results in reactivation of epigenetically silenced genes. In conclusion, this work provides new insights how TET proteins can set DNA modifications, how these oxidized bases are read by various factors and how TET proteins can be posttranslationally modified. Furthermore, removal of 5mC is achieved through TET-mediated oxidation and depends on the activity of specific glycosylases, which leads to gene reactivation.

Background: Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. Methods: A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. Results: The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual gamma-H2AX foci after 24 h. Conclusion: So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.

Signaling networks control and regulate outcomes in cells and organisms in both normal physiology and pathophysiological states. Signaling is traditionally represented and studied as a series of stepwise enzymatic events constituting a cascade. However, it is increasingly apparent that such representations limit understanding of signal transduction since these linear cascades function in an interconnected network that includes extensive cross talk among receptors and pathways. Mass spectrometry (MS)-based proteomics is a useful tool that allows a system-wide investigation of signaling events at the levels of post-translational modifications (PTMs), protein-protein interactions and changes in protein expression on a large scale. This technology now allows accurate quantification of thousands of proteins and their modifications in response to any perturbation. This thesis work is dedicated to the optimization and employment of quantitative mass spectrometry to cellular signaling and an application to segregate two lymphoma subtypes at the levels of protein expression and phosphorylation, employing state of the art liquid chromatography (LC)-MS/MS technologies coupled with improved sample preparation techniques and data analysis algorithms. In the first project I investigated the feasibility of a new, high accuracy fragmentation method called higher energy collisional dissociation (HCD) for the analysis of phospho-peptides. Using this method we were able to measure the phospho-proteome of a single cell line in 24h of measurement time which was a great improvement to previous capabilities. This fragmentation method that was originally thought to be slower and less sensitive than the standard method of low resolution collision induced dissociation (CID) fragmentation. However, our work proves this not to be the case and we showed that HCD outperformed the existing low resolution strategy [1]. In the second project I employed this HCD fragmentation technique on the LTQ-Orbitrap Velos for addressing the clinical question of segregating two subtypes of diffuse B-cell lymphoma (DLBCL). These subtypes are histologically indistinguishable but had been segregated on the basis of a gene expression signature. I employed the recently developed ‘super-SILAC’ approach with a ‘super-SILAC mix’ of multiple labeled cell lines. This heavy reference mix was spiked into several cell lines derived from the two DLBCL subtypes and analyzed LC-MS, resulting in successful segregation based on a distinct proteomic signature [2]. The third project deals with the in-depth analysis of the phospho-proteome of a human cancer cell line on a quadrupole-Orbitrap mass spectrometer using a label-free quantification approach. Our analysis uncovered about 50,000 distinct phosphorylated peptides in a single cell type across a number of cellular conditions allowing assessment of global properties of this large dataset. Strikingly, we found that at least three-quarters of the proteome can be phosphorylated which is much higher than current estimates. We also analyzed phosphotyrosine events using enrichment with anti-phospho-tyrosine antibodies to identify more than 1,500 site specific phosphorylation events. Unexpectedly tyrosine phosphorylated proteins were enriched among higher abundance proteins. The observed difference in phospho-protein abundance correlated with the substrate Km values of tyrosine kinases. For the first time we calculated site specific occupancies using label- free quantification and observed widespread full phosphorylation site occupancy during mitosis. In the final and main project, I applied proteomics and phospho-proteomics to the study of signal transduction in response to transforming growth factor-beta (TGF-β), a multifunctional cytokine. TGF-β signaling regulates many biological outcomes including cell growth, differentiation, morphogenesis, tissue homeostasis and regeneration. The cellular responses to this multifunctional ligand are diverse and can even be opposed to each other, depending on the cell type and the conditions. To shed light on the reasons for the different outcomes, we analyzed the early phospho-proteome and ensuing proteome alterations in response to TGF-β treatment in a keratinocyte cell line. The early SILAC based phospho-proteome analysis uncovered over 20,000 phosphorylation events across five time points (0 to 20 min) of TGF-β treatment. Building on our recent advances in instrumentation, sample preparation, and data analysis algorithms we measured a deep TGF-β responsive proteome at six late time points (6h to 48h) with corresponding controls in only eight days of measurement time. Our label-free approach identified about 8,000 proteins and quantified more than 6,000 of them. This deep proteome covered well established pathways involved in TGF-β signaling, allowing global evaluation at the level of individual pathway members. Combining the TGF-β responsive proteome with an in-silico upstream regulator analysis, we correctly retrieved several known and predicted novel transcription factors driving TGF-β induced cytostasis, de-differentiation and epithelial to mesenchymal transition (EMT). The combined analysis of transcription factor regulation with early phosphorylation changes and proteome changes enabled visualization of the intricate interplay of key transcription factors, kinases and various pathways driving cytostatis, EMT and other processes induced by TGF-β. In summary, my thesis developed a highly efficient phospho-proteomic workflow, which was applied to the measurement of a very deep phospho-proteome of a single cancer cell line allowing analysis of its global features. The main achievement was the first in-depth and combined study of the phospho-proteome and resulting proteome changes following a defined signaling event, in this case leading to a time-resolved view of TGF- β signaling events relevant in cancer.

Posttranslational modifications (PTMs) of proteins by covalent attachment of functional groups (like phosphorylation, acetylation, methylation, glycosylation, etc.) are of key importance for the cell as they regulate various aspects of protein behavior after its synthesis, e.g., dictate protein interaction properties, change catalytic activity of enzymes, induce conformational changes, guide subcellular localization and determine protein stability. A special class of protein PTMs is the conjugation of small proteins of the ubiquitin family to typically acceptor lysine residues of the substrates. The reversible nature of this PTM and the presence of dedicated domains that specifically recognize modified substrates make this type of protein modification instrumental for the regulation of numerous biological pathways. For ubiquitylation, strong substrate selectivity due to the presence of highly diversified conjugation machinery is characteristic and well studied, especially in case of ubiquitin’s proteolytic role. On the contrary, much less is known about the principles of substrate specificity and mechanisms of PTM action in the ubiquitin-like protein SUMO modification system. Despite the fact that SUMOylation specifically targets hundreds of substrates and major conjugation steps are identical with ubiquitin system, strikingly only a handful of enzymes operate in the SUMO pathway, suggesting that other principles of substrate selectivity must apply and perhaps distinct mechanisms of PTM action exist in the SUMO pathway. Moreover, the recognition of SUMO modification is surprisingly simple and relies mainly on a short hydrophobic sequence known as SUMO-interacting motif (SIM), in striking contrast to the ubiquitin system, where numerous ubiquitin-binding domains exist with different interaction specificities. All these, together with the observations that SUMO conjugation machinery seems rather promiscuous in vitro, that typically only a small fraction of a protein is being SUMOylated at a given time, and that specific SUMOylation-defective mutants often exhibit no obvious phenotypes, whereas SUMO pathway mutants do, emphasize the question of substrate specificity in the SUMO system and suggest other principles of SUMO action on its substrates. Here, we address the question of SUMOylation specificity and function using DNA double-strand break (DSB) repair pathway via homologous recombination (HR) as a case study because of its strong ties to the SUMO system. First, using SILAC-based proteomic approach we show that proteins acting in the same DNA repair pathway become collectively SUMOylated upon a specific stimulus (HR factors – upon DSB induction; nucleotide excision repair factors – upon exposure to UV light), suggesting that SUMO machinery often targets protein groups within the same pathway. Then, focusing on the DSB repair we find that DNA-bound SUMO ligase Siz2 catalyzes collective multisite SUMOylation of a whole set of HR factors. Repair proteins are loaded onto resected single-stranded DNA (ssDNA) in the vicinity of the ligase, thus making exposure of ssDNA a precise trigger for modification. Protein group SUMOylation fosters physical interactions between the HR proteins engaged in DNA repair, because not only that they become collectively modified at multiple SUMO-acceptor sites, but they also possess multiple SIMs, which promote SUMO-SIM mediated complex formation. Only wholesale elimination of SUMOylation of the core HR proteins significantly affects the HR pathway by slowing down DNA repair, suggesting that SUMO acts synergistically on several proteins. Thus, we show that SUMOylation collectively targets functionally engaged protein group rather than individual proteins, whereas localization of modification enzymes and specific triggers ensure substrate specificity.

Desoxyribonuklerinsäure (DNA), die Erbinformation, definiert die Struktur und Funktion jeder Zelle. In Eukaryoten ist sie um Oktamere aus den Histonen H2A, H2B, H3 und H4 gewunden. Sie bilden mit der DNA höhere Strukturen, das sog. Chromatin. Die Struktur des Chromatins beeinflusst direkt die Aktivität der gebundenen DNA. Eukaryoten besitzen daher viele molekulare Mechanismen zu ihrer Veränderung, z.B. posttranslationale Modifizierungen (PTMs) von Histonproteinen oder den Austausch von kanonischen Histonen mit Histonvarianten (z.B. H3.1, H3.2, H3.3, u.a.). Für einige Varianten sind ihnen eigene, charakteristische PTMs beschrieben, z.B. die Phosphorylierung des Serins 31 der Variante H3.3 (H3.3S31ph). Die Histone Code Hypothesis postuliert, dass PTMs von Histonen in festen Mustern vorliegen können. Die Switch Hypothesis beschreibt die Regulation von Bindemolekülen an Histone durch benachbarte PTMs. Auf ihrer Grundlage wurde die These der Doppelmodifizierung einer bekannten Trimethylierung der Aminosäure (AS) Lysin 79 mit einer mutmaßlichen Phosphorylierung der AS Threonin 80 auf Histon H3 aufgestellt (H3K79me3T80ph). Neben der Etablierung eines einfachen Systems zur Identifizierung bisher unbeschriebener Phosphorylierungen bestand ein zweites Ziel dieser Arbeit im Nachweis der Phosphorylierung von H3 Threonin 80 in vivo. Eine weitere Zielsetzung lag in der genaueren Charakterisierung der bereits beschriebenen Varianten-spezifischen PTM H3.3S31ph, deren Expression zwar eng umschrieben ist, über deren spezifische Funktionen, Kinase und mögliche Effektorproteine aber wenig bekannt ist. Um einen Überblick über Phosphorylierungen verschiedener Histonroteine zu gewinnen, wurden unterschiedliche Polyacrylamidgel-Elektrophoresen Verfahren (PAGE) etabliert. Zum Einsatz kamen A/U-, T/A/U- und 2D-T/A/U-PAGE Verfahren. Sie ermöglichten in Übereinstimmung mit der Literatur die Auftrennung bekannter PTM und stehen nun für weiterführende Studien zur Verfügung. Der massenspektrometrische Nachweis der putativen PTM H3K79me3T80ph in vivo gelang nicht. Trotz optimierter Versuchsbedingungen konnte die Phosphorylierung weder in der MALDI-ToF, noch in der Orbitrap MS/MS nachgewiesen werden. Initiale Antikörperdaten wurde aufgrund einer aufgedeckten Kreuzreaktivität in Frage gestellt. Obgleich nicht mit letzter Sicherheit gesagt werden kann, dass H3K79me3T80ph in vivo nicht existiert, wurde die These letztlich verworfen. Die molekularbiologische Untersuchung der Varianten-spezifischen PTM H3.3S31ph ergab bei verifizierter Überexpression und Chromatin-Integration punktmutierter Histone übereinstimmend Hinweise auf einen Effekt der PTM auf die Zellteilung. Es konnte gezeigt werden, dass Serin 31 bzw. ihre PTM H3.3S31ph sowohl Zellzyklus, als auch Wachstums- und Proliferationsgeschwindigkeiten von HeLa Zellen beeinflusst. Dies argumentiert für eine aktivierende Funktion von H3.3S31ph in der Mitose. Weiter konnten mithilfe eines ELISAs fünf potentielle Proteinkinasen für H3.3S31ph identifiziert werden. Vorrangig kommt dabei die nukleäre Kinase PIM1 in Betracht. Zur weiteren Untersuchung potentieller Effektorproteine wurde in vitro ein molekularbiologisches Modellsystem etabliert. Es steht nun für weiterführende Studien zur Verfügung. Erste Vorarbeiten konnten bereits zeigen, dass hier evtl. Interaktionen mit dem Linkerhiston H1 eine wichtige Rolle spielen.

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state.

Special Issue "Animal models in human disease", Molecular chaperone function in sickle red cells, Reports, PTMs during differentiation in Leishmania, A protein kinase C alpha-specific substrate, Viewpoint Interview: Deja vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins

Special Issue "Animal models in human disease", Molecular chaperone function in sickle red cells, Reports, PTMs during differentiation in Leishmania, A protein kinase C alpha-specific substrate, Viewpoint Interview: Deja vu in proteomics. A hit parade of repeatedly identified differentially expressed proteins