Periodic Reporting for period 4 - Ubl-Code (Revealing the ubiquitin and ubiquitin-like modification landscape in health and disease)
Período documentado: 2020-11-01 hasta 2021-10-31
EpiProteomics, the regulation of post-translational modifications (PTM) and protein degradation, underlie key aspects of protein function and are crucial for maintaining cellular proteostasis. Our lab uses mammalian systems (in vitro and in vivo) to explore the PTM- and degradation- dependent vulnerabilities of cells and decipher the underlying mechanisms in the context of cancer and immunity.
Understanding the spatio-temporal control of PTMs and the role of proteasome heterogeneity and degradation requires challenging analytical systems. The power of our lab is the interdisciplinary capabilities, bridging biochemistry, cell biology, immunology and in vivo models together with proteomics & computational tools, that enable us to develop novel proteomics-based approaches to transform capabilities in epiProteomics and precision medicine. Over the past 6 years our efforts have been dedicated to the development of novel technologies including three distinct platforms to: 1) globally investigate enzymatic modification activity in primary biological specimens, 2) directly analyze proteasome degradation products, and combine the knowledge of these regulatory level to bring insight into the pathophysiology of human disease. We identified novel mechanisms of regulation in the ubiquitin-proteasome system that may translate into therapeutic interventions.
Understanding the spatio-temporal control of PTMs and the role of proteasome heterogeneity and degradation requires challenging analytical systems. The power of our lab is the interdisciplinary capabilities, bridging biochemistry, cell biology, immunology and in vivo models together with proteomics & computational tools, that enable us to develop novel proteomics-based approaches to transform capabilities in epiProteomics and precision medicine. Over the past 6 years our efforts have been dedicated to the development of novel technologies including three distinct platforms to: 1) globally investigate enzymatic modification activity in primary biological specimens, 2) directly analyze proteasome degradation products, and combine the knowledge of these regulatory level to bring insight into the pathophysiology of human disease. We identified novel mechanisms of regulation in the ubiquitin-proteasome system that may translate into therapeutic interventions.
Ubiquitin and ubiquitin-like protein modifications and PTM profiling:
We developed the first systematic ubiquitination assay in liquid biopsies and showed that ubiquitination signatures differentiate between healthy and autoimmune-diseased individuals (i.e. lupus). We provided a proof-of-concept for the ability of our approach to serve for molecular and clinical profiling and biomarker discovery (Exp Biol and Med, 2016, Methods in Mol Biol, 2017).
We discovered that the Small Ubiqiuitn-like MOdifier (SUMO) plays a role in organization of the chromatin landscape to control the developmental trajectory and prohibit reversal of the developmental program. Identified a novel mechanism regulating chromatin organization by SUMOylation of histone H1. The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultracondensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity. (Sheban et al, Molecular Cell. https://doi.org/10.1016/j.molcel.2021.11.011(se abrirá en una nueva ventana)).
By analyzing Sites of Alternative Modifications (SAM) between ubiquitin or SUMO we uncovered properties such as increased abundance and dual cyto-nuclear localization that were associated with SAM-containing proteins. Protein modification by ubiquitin or SUMO can alter the function, stability or activity of target proteins. Previous studies have identified thousands of substrates that were modified by ubiquitin or SUMO on the same lysine residue. However, it remains unclear whether such overlap could result from a mere higher solvent accessibility, whether proteins containing those sites are associated with specific functional traits, and whether selectively perturbing their modification by ubiquitin or SUMO could result in different phenotypic outcomes. Here, we mapped reported lysine modification sites across the human proteome and found an enrichment of sites reported to be modified by both ubiquitin and SUMO. The analysis uncovered thousands of proteins containing such sites, which we term. Among more than 36,000 sites reported to be modified by SUMO, 51.8% have also been reported to be modified by ubiquitin. Comparing the biological and biochemical properties of sites of alternative modification versus other non-overlapping modification sites revealed that these sites were associated with altered cellular localization or abundance of their host proteins. Using S. cerevisiae as model, we could show that mutating the SAM motif in a protein can influence its ubiquitination as well as its localization and abundance. (Ulman A, et al. JMB, Vol. 433, no. 21 10-2021. https://doi.org/10.1016/j.jmb.2021.167219(se abrirá en una nueva ventana)).
Cellular mechanisms of proteasome-dependent degradation and antigen presentation:
We discovered Golgi localized proteasomes that play a role in maintaining Golgi homeostasis by promoting proteasome-dependent degradation of proteins that tether the Golgi cisternae under stress, leading to Golgi fragmentation (Nature Communications, 2020). Our discovery reveals a novel pathway for quality control of the secretory network and is expected to yield a new understanding of human pathologies that are associated with Golgi fragmentation.
We developed mass spectrometry (MS) analysis of proteolytic peptides (MAPP) as the first approach for unbiased profiling of active degradation in single-peptide resolution by analyzing the flux through cellular proteasomes (Nature Biotechnology, 2018, Expert Rev in Prot, 2019).
We could further show that pro-inflammatory cytokines alter the immunopeptidome landscape by modulation of HLA-B Expression in NSCLC (Javitt et al, Frontiers in Immun, 2019).AIM 1 - Mapping the ubiquitin and ubiquitin-like modification landscape in cancer
We developed the first systematic ubiquitination assay in liquid biopsies and showed that ubiquitination signatures differentiate between healthy and autoimmune-diseased individuals (i.e. lupus). We provided a proof-of-concept for the ability of our approach to serve for molecular and clinical profiling and biomarker discovery (Exp Biol and Med, 2016, Methods in Mol Biol, 2017).
We discovered that the Small Ubiqiuitn-like MOdifier (SUMO) plays a role in organization of the chromatin landscape to control the developmental trajectory and prohibit reversal of the developmental program. Identified a novel mechanism regulating chromatin organization by SUMOylation of histone H1. The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultracondensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity. (Sheban et al, Molecular Cell. https://doi.org/10.1016/j.molcel.2021.11.011(se abrirá en una nueva ventana)).
By analyzing Sites of Alternative Modifications (SAM) between ubiquitin or SUMO we uncovered properties such as increased abundance and dual cyto-nuclear localization that were associated with SAM-containing proteins. Protein modification by ubiquitin or SUMO can alter the function, stability or activity of target proteins. Previous studies have identified thousands of substrates that were modified by ubiquitin or SUMO on the same lysine residue. However, it remains unclear whether such overlap could result from a mere higher solvent accessibility, whether proteins containing those sites are associated with specific functional traits, and whether selectively perturbing their modification by ubiquitin or SUMO could result in different phenotypic outcomes. Here, we mapped reported lysine modification sites across the human proteome and found an enrichment of sites reported to be modified by both ubiquitin and SUMO. The analysis uncovered thousands of proteins containing such sites, which we term. Among more than 36,000 sites reported to be modified by SUMO, 51.8% have also been reported to be modified by ubiquitin. Comparing the biological and biochemical properties of sites of alternative modification versus other non-overlapping modification sites revealed that these sites were associated with altered cellular localization or abundance of their host proteins. Using S. cerevisiae as model, we could show that mutating the SAM motif in a protein can influence its ubiquitination as well as its localization and abundance. (Ulman A, et al. JMB, Vol. 433, no. 21 10-2021. https://doi.org/10.1016/j.jmb.2021.167219(se abrirá en una nueva ventana)).
Cellular mechanisms of proteasome-dependent degradation and antigen presentation:
We discovered Golgi localized proteasomes that play a role in maintaining Golgi homeostasis by promoting proteasome-dependent degradation of proteins that tether the Golgi cisternae under stress, leading to Golgi fragmentation (Nature Communications, 2020). Our discovery reveals a novel pathway for quality control of the secretory network and is expected to yield a new understanding of human pathologies that are associated with Golgi fragmentation.
We developed mass spectrometry (MS) analysis of proteolytic peptides (MAPP) as the first approach for unbiased profiling of active degradation in single-peptide resolution by analyzing the flux through cellular proteasomes (Nature Biotechnology, 2018, Expert Rev in Prot, 2019).
We could further show that pro-inflammatory cytokines alter the immunopeptidome landscape by modulation of HLA-B Expression in NSCLC (Javitt et al, Frontiers in Immun, 2019).AIM 1 - Mapping the ubiquitin and ubiquitin-like modification landscape in cancer