23rd Human Proteome Organization World Congress

Prof. Dr.

Global organelle profiling reveals subcellular localization and remodeling at proteome scale

Keynote Lecture

Defining the subcellular distribution of all human proteins and its remodeling across cellular states remains a central goal in cell biology. Here, we present a high-resolution strategy to map subcellular organization using organelle immuno-capture coupled to mass spectrometry. We apply this workflow to a cell-wide collection of membranous and membrane-less compartments. A graph-based analysis reveals the subcellular localization of over 7,600 proteins, defines spatial networks, and uncovers interconnections between cellular compartments. Our approach can be deployed to comprehensively profile proteome remodeling during cellular perturbation. By characterizing the cellular landscape following hCoV-OC43 viral infection, we discover that many proteins are regulated by changes in their spatial distribution rather than by changes in abundance. Our results establish that proteome-wide analysis of subcellular remodeling provides unique insights for the elucidation of cellular responses, uncovering an essential role for ferroptosis in OC43 infection. Our dataset can be explored at organelles.czbiohub.org.

Keynote lecture

Prof.

Proteomic translation and degradation screen of HECT E3 ligases reveals functional redundancies and exclusivity

Cell Biology Insights

Homologous to E6AP C-terminus (HECT) E3 ubiquitin ligases are crucial for maintaining cellular proteostasis by regulating ubiquitination and subsequent degradation of substrates. These enzymes are integral to various biological processes, including protein trafficking, subcellular localization, and immune responses. Defects in HECT E3 ligase functionality have been implicated in various cancers, neurodegenerative diseases, and viral infections.With the advent of PROteolysis TArgeting Chimaera (PROTAC) technology, HECT E3 ligases have emerged as promising targets for therapeutic intervention. Despite the wide range of pharmacological possibilities, only a subset of the 28 annotated human HECT E3 ligases have been studied in depth. Consequently, much remains unknown about their substrates and regulatory mechanisms.To address this, we performed a high-throughput translation/degradation proteomic screen coupled with siRNA-induced knockdown of 27/28 HECT E3 ligases. Expanding on our previously established multiplexed enhanced protein dynamic (mePROD) approach, we combined an siRNA screen with pulsed stable isotope labelling in cells (pSILAC) and isobaric tagging (TMT) to temporally quantify protein degradation and synthesis for each HECT E3 ligase upon knockdown. We were able to quantify &gt;8,000 proteins/siRNA in both translation and degradation proteoform states from a single well of a 96-well cell culture plate. Moreover, knockdown was confirmed for the majority of siRNA targets in proteomics data, further increasing the confidence in the efficacy of our optimized high-throughput experimental approach.Seventeen out of 27 siRNAs showed no drastic difference in overall protein translation and degradation or cell viability, suggesting a narrow range of downstream modules and functional synergism, with a few unique set of differentially regulated proteins corresponding to each HECT E3 ligase. Ten out of 27 siRNAs severely impacted the overall proteome turnover, with a broad effect on protein translation and degradation, suggesting exclusive dependence on these specific HECT E3 ligases and their crucial role in the regulation of cellular proteostasis.UBR5 has previously been reported to regulate a large number of transcription factors and nuclear proteins. Our data corroborates this, with evidence of reduced protein degradation and cell viability. Similarly, our data confirm known modalities in HECTD2-4, NEDD4, HUWE1 and provides evidence for novel functional substrates with potentially wide therapeutic relevance ranging from cancer, cardiac hypertrophy, inflammation to antidepressants.In conclusion, we provide here a high-throughput approach to study protein synthesis and degradation that can be combined with siRNA, CRISPR and compound screens. Our findings provide a valuable resource for understanding HECT-mediated protein degradation and paves the way for drug design in various therapeutic contexts.

Oral presentation

Divergent proteome tolerance against gain and loss of chromosome arms

Organisation of the Proteome (PPI)

Aneuploidy, defined as the abnormal gain or loss of chromosomes or chromosome arms, causes human genetic syndromes and many cancers. For example, in lung squamous cell carcinoma, chromosome 3q is gained in over 60% of tumors, while chromosome 3p loss occurs in 78% of tumors. However, how the cellular proteome tolerates the gain or loss of chromosomes—a fundamental question in aneuploidy research—remains controversial. We and others have previously reported that aneuploid eukaryotic cells enhance the dynamic degradation of unbound protein complex subunits encoded by excessive chromosomes, maintaining protein complex stoichiometry for cellular biochemical reactions. This mechanism was recently speculated to occur in both gain and loss-type aneuploidies. However, this means that loss-type aneuploid cells would have to degrade all proteins not encoded by the aneuploid (deficient) chromosome but participating in the same protein complexes to a much lower level, which is not economic for the proteomic organization.In the present work, using a CRISPR engineering strategy, we generated an isogenic chromosome 3 isogenic aneuploidy model exhibiting either 3p loss or 3q gain in lung epithelial cells, along with euploid controls. We then performed a systems analysis of protein abundance, turnover, and thermal stability using mass spectrometry (MS) technologies, including data-independent acquisition (DIA)-MS quantification, dynamic SILAC or pulse SILAC (pSILAC) labeling with plex-DIA, and cellular thermal shift assays (CETSA), on a series of isogenic cells. Our plex-DIA data, based on nearly 9,000 proteins, remarkably found no changes in protein degradation rate for complex-in or complex-out 3p proteins in 3p loss cells, demonstrating that deficient gene copies due to chromosomal loss do not entail slower protein degradation. Instead, we discovered that protein dosage compensation is tightly associated with a relatively enhanced synthesis rate of 3p-encoded proteins participating in protein complexes. Additionally, the interaction partners of proteins encoded by duplicated 3q or deleted 3p are both pervasively regulated post-transcriptionally (mRNA-protein correlations are barely 0.047~0.13). CETSA results further show that proteins encoded on deleted 3p exhibit higher thermal stability, implying the presence of stronger protein-protein interactions. In stark contrast, thermal stability is not regulated for 3q proteins in 3q gain cells. Finally, to corroborate the tolerance mechanism of selective protein translation in lost-type aneuploidy, we found that cytosolic ribosomal proteins, although downregulated, are dramatically stabilized at the post-translational level through longer protein lifetimes and higher thermal stability in 3p loss cells, suggesting a fine-tuning of the protein synthesis process.In summary, our results demonstrate that cells utilize distinct proteome response mechanisms to tolerate the gain and loss types of aneuploidies.

Professor

Mapping communication dynamics between physically interacting cells using HySic

New Technology: MS-based Proteomics

Summary. Cytotoxic CD8+ T cells engage their targets and form physical connections that drive the release of cytotoxic compounds, thus playing a crucial role in anti-cancer immunity. Yet, our full understanding of the proteins involved in this process remains elusive. To study the mechanisms underlying tumor resistance to T cell attack,ar events, we have developed HySic (hybrid quantification of stable isotope labeling by amino acids in cell culture [SILAC]-labeled interacting cells), a method to quantify short-term protein and phosphorylation dynamics between and within physically-interacting cells (PMID: 38150364). We benchmarked our method by analysing the communication between activated primary CD8+ T-cells and non-small-cell lung cancer cell lines over a time course of 0-6 hours.Method. First, we incorporated heavy SILAC labels into cultured tumor cells. Then, we co-cultured them with primary activated T cells in media containing intermediate SILAC label. All cells were harvested together and we used label-free quantification (LFQ) to quantify protein and phosphorylation dynamics in T-cells (light SILAC), tumor cells (heavy SILAC) and in newly-synthesized proteins (intermediate SILAC). Next, we sought to increase the throughput and sampling depth of our HySic method by replacing LFQ quantification with multiplexed TMT combined to FAIMS. Here, each co-culture timepoint was tagged with a different TMT channel. After sample pooling and fractionation, we employed real-time-searching (RTS) to trigger MS3-based TMT quantification. Using their respective SILAC label, each target population (tumor, T-cell or newly-synthesized proteins) can be enriched with RTS.Results. In total HySic quantified 2742 proteins and 7005 phosphosites in T-cells, and 5469 proteins and 19428 phosphosites from tumors while HySic-TMT led to the quantification of 2.5-times more proteins in 2 times less runtime. Enrichment analysis of T-cell proteome data yielded leukocyte-specific terms, indicating selective quantification of T-cells in a mixed proteome population. We observed specific downregulation of granulin in tumor cells and CD8 in T-cells, which were both validated by additional methods. In our phosphoproteome data, we observed that following engagement, both T-cells and tumors upregulate Rho-GTPase signaling through PAK1 activation. By applying a PAK1 inhibitor, we were able to increase the migration and the production of cytotoxic granzyme-B by T cells, which resulted in increased tumor killing.Significance. Altogether, our results demonstrate the ability of HySic to identify actionable targets that improve immune-mediated tumor clearance. Moreover, implementing TMT-RTS-MS3 in the HySic workflow opens the possibility to quantify large experimental datasets of mixed-cell signaling dynamics with minimal instrumentation time. We demonstrated that our HySic method is an innovative and efficient approach to probe signaling dynamics between interacting cells.

Conference room 1-2

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Proteome dynamics

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Proteome dynamics

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SessionProteome dynamics

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Vorsitzende

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Session
Proteome dynamics