UbiCODE network-wide training. The participation of institutions in the meetings, courses and workshops is indicated. Lead institutions are indicated in Table 1.2 b. Our network-wide training is a transversal program providing support beyond our four scientific work packages by preparing ESRs to face their career in public research institutions or the private sector.


SUMOylation and Ubiquitylation

Duration : 5 days
Lead Institution : IMB (Ulrich)
Project Month : January 2019

WP1-3. Collaborators: MPGa (Pichler), GUF (Dikic), MRC-PPU (Kulhatu), MPGb (Tirard), UNILIV (Urbé)

Objective: To introduce ESRs to the enzymology of the Ub and SUMO systems, the principles of Ub and SUMO chain synthesis, biochemistry and recognition.
Abstract: This course will provide a theoretical and practical introduction to the principles of SUMO and Ub conjugation, chain formation and recognition in vitro and in vivo. It will cover the relevance of chains for Ub and SUMO signalling and the significance of linkage specificity in the synthesis and recognition of Ub chains. ESRs will learn how to conduct ubiquitylation and SUMOylation reactions, activity and interaction assays, design appropriate controls and avoid common pitfalls.
Day 1: Overviews from PIs.
Day 2: Conjugation enzymes & assays; E1 & E2: thioester assays and chain formation; E3: lysine discharge assays; E3: substrate-specific modification.
Day 3: SUMO and Ub binding and linkage specificity; Linkage-specific E3s; in vitro analysis of UBDs and SIMs; Y2H assays for analysis of UBDs and SIMs.
Day 4: Deconjugation enzymes & assays; DUB assays; DUBs as tools for Ub linkage analysis.
Day 5: Detection of Ub and SUMO substrates; Target identification; In vivo analysis of Ub and SUMO targets.


Cell biological methodologies to study protein ubiquitylation

Duration : 4 days
Lead Institution :UNILIV (Urbé)
Project Month : June 2019

WP1-4. Collaborators: UPV/EHU (Mayor)

Objective: To introduce ESRs to a wide range of cell biological methodologies with a focus on the role of reversible ubiquitylation in endocytic trafficking and selective autophagy in mammalian cells.
Abstract: This course will provide valuable grounding in cell biological considerations to all participants. We will introduce ESRs to a wide range of cell biological methodologies with a focus on the role of reversible ubiquitylation in endocytic trafficking and selective autophagy in mammalian cells. The course will provide hands-on experience with live cell imaging and image analysis of transiently transfected and stable cell lines, subcellular fractionation techniques and include theoretical introductions to siRNA and CRISPR-based KO technologies.
Day 1: Mammalian cell culture; Transfection; Seminars on RNAi and CRISPR KO technology/screens; Live cell imaging.
Day 2: Fixation and staining of cells; Subcellular fractionation.
Day 3: Image analysis of live cell imaging experiments; Imaging of fixed cells; Western blot analysis of subcellular fractions.
Day 4: Image analysis and presentations.


Crosstalk between Ub family members under stress conditions

Duration : 5 days
Lead Institution : CNRSa (Rodriguez)
Project Month : June 2019

WP2, WP3. Collaborators: CNRSb (Xirodimas), CNRSc (Lutz), CNRSd (Sumara)

Objective: To train ESRs on techniques for detection of total and individual proteins simultaneously modified by Ub and UbLs.
Abstract: Detection of proteins modified by distinct members of the Ub family is still a challenge. Distinct methods will be used to analyse Ub chain composition including detection of Ub-NEDD8 and Ub-SUMO hybrid chains and their recognition by distinct receptors. Biochemical outcomes from Course 1 will be considered to explore biological responses in stressed cells. Total or individually modified proteins will be detected using chain specific minibodies or antibodies to detect proteins of interest and also by MS. Day 1: Theory of the analysis of complex hybrid chains; Use of MS approaches; Treatment of cells with distinct stresses (e.g. proteasome inhibitors, ATO).
Day 2: Capture of proteins modified with Ub-NEDD8 using Ub and NEDD8 traps; Use of inhibitors of UBD-Ub interactions; Western-blot analysis with various antibodies; Prepare samples for MS analysis.
Day 3: Theory of the analysis of determining affinities of Ub, NEDD8, SUMO receptors; Treatment of cells; Use of inhibitors of UBD-Ub interactions.
Day 4: Capture of proteins modified with Ub-SUMO using distinct molecular traps; Western-blot analysis with various antibodies; Preparation of samples for MS analysis.
Day 5: Data analysis and interpretation; Final discussion.


Ubiquitinome analysis

Duration : 5 days
Lead Institution : GUF (Dikic)
Project Month : June 2019

WP1-4. Collaborators: GUF (Marculis)

Objective: To train ESRs in the analysis of ubiquitinome.
Abstract: This workshop will introduce proteomics techniques for studying ubiquitylation processes. The methods conveyed will include substrate identification techniques, interactome studies and determining the global ubiquitinome. ESRs will acquire hands-on experience in designing and conducting experiments for deciphering ubiquitylation processes in depth, including sample preparation, MS run and conclude with an extensive analysis and interpretation of the results. A Q&A session will focus on pitfalls and troubleshooting of proteomic approaches.
Day 1: Overview and introduction: Technology platform; MS: Get to know about machines, methods, and analysis of results; Ub Interactome: Immunoprecipitation of Ub and/or Ub binding proteins and analysis of co-immunoprecipitating proteins and MS analysis.
Day 2: Substrate identification: Immunoprecipitation of DUB or E3 ligase (inhibited or catalytic dead) followed by determination of associated and accumulated proteins (SILAC labelled cells) and MS analysis.
Day 3: Ubiquitinome analysis (DiGly approach): Treatment of SILAC-labelled cells, immunoprecipitation of DiGly peptides (remnant on ubiquitylated peptides after cleavage for MS) and MS analysis.
Day 4: Linear ubiquitinome.
Day 5: Data analysis (MaxQuant); Discussion of results; Troubleshooting/False positive/Pitfalls of the MS.


Insight day into pharmaceutical Drug Discovery

Duration : 0,5 day
Lead Institution : Merck (Dolt)
Project Month : June 2019


Objective: Give ESRs insights into Pharma industry Drug Discovery & development.
Abstract: Merck will provide one insight day into pharmaceutical Drug Discovery and development, emphasising oncology. In addition to presentations, selected laboratories will be visited to get an insight into industrial research infrastructure. A bus tour around the Darmstadt premises will be organised.
Day 1: Pharmaceutical Drug Discovery & development; Campus bus tour; Lab tour.


Peptide and protein synthesis

Duration : 3 day
Lead Institution : LUMCb (Ovaa)
Project Month : January 2020

WP1-4. Collaborator: LUMCa (Vertegaal)

Objective: To train ESRs to optimally use chemical options in their research.
Abstract: This theoretical and practical course aims to teach ESRs the basics of peptide and protein chemistry. The course precedes a proteomics course to experiment with chemical probes generated in this course. The course is of interest to all participants from all WPs from both academia and industry.
Day 1: Basics of peptide and protein chemistry: Introduction to basic organic chemistry and techniques used with special emphasis on peptide chemistry and protein modification.
Day 2: Practical probe chemistry: ESRs will generate their own activity-based probes tailored to meet their own needs. Techniques used include automated synthesis, LC-MS, HPLC, SDS-PAGE, fluorescent scanning.
Day 3: Practical session: labelling targets in cell lysates. ESRs will label cell extracts of interest with probes generated the prior day. Techniques used include SDS-PAGE and fluoresce scanning. Targets from known inhibitors will be determined to demonstrate the use of activity-based probes in Drug Discovery. Samples will be prepared for advanced MSMS analysis in the ensuing course organised by LUMCa.


Mass spectrometry

Duration : 5 days
Lead Institution : LUMCa (Vertegaal)
Project Month : January 2020

WP1-4. Collaborators: UNIVDUN (Hay), NOVA (Matthiesen)

Objective: To train ESRs on dissecting signal transduction by Ub and UbL proteins. Abstract: The unbiased identification of Ub and UbL target proteins and their conjugation and deconjugation machineries is key for understanding Ub and UbL signal transduction. Following protein purification procedures using His-tagged molecules, unbiased identification of the enriched proteins is carried out by MS. Protein samples are digested by trypsin, peptide mixtures are separated by chromatography and peptides are sequenced by tandem MS. Raw data are processed to carry out protein identification and bioinformatics enables insight in the large datasets obtained.
Day 1: Purification of Ub target proteins; Enrichment strategies for Ub target proteins. Day 2: Purification of UbL target proteins; Enrichment strategies for UbL target proteins.
Day 3: MS; High pressure nano liquid chromatography and Orbitrap MS.
Day 4: Bio-informatics; Processing of raw MS data using MaxQuant software.
Day 5: Bio-informatics; Preparation of overview figures using Perseus software.


Understanding Ub-protein interactions at the molecular level

Duration : 4 days
Lead Institution : MRC-PPU (Kulathu)
Project Month Duration : January 2020

WP1-4. Collaborator: FMI (Thomä)

Objective: To train ESRs on protein biochemistry and on how to gain molecular insights into the enzymes and proteins of the Ub system.
Abstract: Ub interactions are at the core of the Ub system, and their understanding at the molecular level has propelled the field forward. In this course, ESRs will learn about the latest protein biochemistry techniques and methods to make Ub and study Ub interactions with biophysical and structural methods. The course will enable ESRs to understand the structural data available, and teach them what is needed to obtain similar data for their project.
Day 1: Introduction by all teachers, introduction of ESR projects.
Day 2: Theoretical: protein biochemistry, requirements for successful biophysics and structural studies, options for studying Ub binding, introduction to NMR; Practical: measuring Ub interactions by NMR.
Day 3: Theoretical: quantitative methods to measure Ub-protein interactions, introduction to fluorescence, FP, ITC, SPR. Introduction enzyme kinetics; Practical: Biophysics: measuring KD by FP and ITC, measuring DUB kinetics by FP.
Day 4: Theoretical: X-ray crystallography overview and introduction; how good is a structure? What can I do with it?; Practical: Handling protein structures with Pymol.


Communication & Outreach Strategies

Duration : 3 days
Lead Institution : The Naked Scientists (Smith)
Project Month : June 2020


Objective: To train ESRs on public engagement.
Abstract: We will train ESRs with relevant and transferable skills that will enable them to significantly increase the impact of their research program amongst the general public and scientific community. Includes training in podcast production, interview techniques, online publishing and the use of social media for scientific dissemination.
Day 1: Podcasts preparation for relevant webpages.
Day 2: Articles writing for publication in scientific journals.
Day 3: Preparation of videos.


Ub system targeted Drug Discovery

Duration : 0,5 day
Lead Institution : Ubiquigent (Brown)
Project Month Duration : June 2020

WP4. Collaborator: Novartis (Renatus)

Objective: To discuss and explore with the ESRs how to define and address the needs of organisations interested in or already engaged in Ub system targeted Drug Discovery.
Abstract: The lectures will cover all steps for Drug Discovery. i) We will discuss the above in the context of how parallel fields – such as kinase focussed Drug Discovery – have been supported in the past and focus specifically on the challenges which Drug Discovery investigators in the Ub field may face and how these may be overcome. ii) For several DUBs it is thought that their inhibition is beneficial for treating human diseases. However, only few successful approaches to develop small molecule DUB inhibitors have been described so far. We will discuss Drug Discovery efforts on two DUB enzymes to highlight specific challenges and show how they were addressed.
Day 1: Key elements of the Drug Discovery process; Target identification and validation; Assay development; Compound screening; Hit identification and profiling; Target engagement and compound testing in therapeutically relevant assays; Discovering inhibitors of DUB enzymes; Small molecules inhibitors; Development of specific DUBs inhibitors.


How to start a biotech company

Duration : 0,5 day
Lead Institution : Hybrigenics (Rain)
Project Month : June 2020


Objective: To demystify the creation of a biotech company among the ESRs of this program to encourage the creation of spin-offs.
Abstract: This course will be developed around the early steps of the company creation with three modules: i) From the idea to the project, how to enrich and idea, from the idea to the costumer value proposition; ii) From the customer value proposition to the business plan; iii) Illustration with biotech companies in the UbiCODE project.
Day 1: Hybrigenics: the first 5 years; UbiQ: A technology drive company in the Ub world.


Grant writing from PhD to Scientific Leadership

Duration : 1 day
Lead Institution : Yellow Research (Jaspers)
Project Month : June 2020

WP1-4. Collaborator: LUMCa (Vertegaal)

Objective: To provide ESRs with strategic input for writing competitive individual grants.
Abstract: We will focus on the importance of drafting the research project for an individual grant. This means that ESRs have to learn a whole new vocabulary: ranging from scientific breakthrough, challenges, aims, objectives, outputs, results to impact. We will make use of best practices developed with (national) and EC funding schemes like the MSC fellowships and ERC in particular.
Day 1: Part1 Outstanding CV: Conceptual framework funding organisations; Overview potential national and EC funding; Highlighting the differences between individual grants and consortium based grants; Personal presentation: Qualities of Principal Investigators; Building a scientific CV: How to present your publication and research track-record, mobility, supervision etc.; Discussions. Part 2 Understanding the Grant Writing vocabulary: From idea to concept visualization; From overarching aim to specific objectives and everything between rationales and impact; Project implementation aspects: WPs, time schedule and risk analysis; Group Work and final discussion.