Project description:Targeted protein degradation (TPD) has emerged as a powerful strategy to selectively eliminate cellular proteins using small-molecule degraders, offering therapeutic promise for targeting proteins that are otherwise undruggable. However, a remaining challenge is to unambiguously identify primary TPD targets that are distinct from secondary downstream effects in the proteome. Here we introduce an approach that combines stable isotope labeling and click-chemistry for selective quantification of protein degradation by mass spectrometry, excluding confounding effects of altered transcription and translation induced by target depletion. We show that the approach efficiently operates at the time scale of TPD (hours) and we demonstrate its utility by analyzing the Cyclin K degraders dCeMM2 and dCeMM4, which induce widespread transcriptional downregulation, and the GSPT1 degrader CC-885, an inhibitor of protein translation. Additionally, we apply it to characterize compound 1, a previously uncharacterized degrader, and identify the zinc-finger protein FIZ1 as a degraded target.
Project description:The relevance of mitochondrial proteostasis for the differentiation and function of hematopoietic and immune cells is unknown. Dissecting the HCLS1-associated protein X-1 (HAX1) –related protein network, we define a functional CLPB-HAX1-PRKD2-HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and thus elucidate molecular mechanisms underlying congenital neutropenia in patients with mutations in HAX1 and CLPB. As shown by SILAC proteomics, CLPB and HAX1 control the balance of protein synthesis and persistence in mitochondria, leading to dysfunction of the respiratory complex 1. Impaired mitochondrial proteostasis is associated with decreased abundance of the serine-threonine kinase PRKD2 and Ser82-phosphorylated HSP27. Cellular defects in HAX1-/- cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical mechanism governing the differentiation and function of neutrophil granulocytes.
Project description:We performed gene expression analysis using Affymetrix U133Plus2 microarray chips on cultures of infected human cells. To mimic an in vivo infection, we infected human monocyte-derived macrophages (MDMs) in vitro with wild-type L. chagasi parasites and then co-cultured those infected MDMs with naive, autologous T-cells.
Project description:Proteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). To address this gap, we employ stable isotope labelling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated and ubiquitinated peptides for >9,000 native proteins. This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of PTMs. While causal relationships may not always be immediately apparent, we hypothesize that PTMs with diverging turnover may distinguish states of differential protein stability, structure, localization, enzymatic activity, or protein-protein interactions. We exemplify how the turnover data may facilitate insights into unknown functions of PTMs and provide a web-tool for the scientific community that allows interrogation and visualisation of all turnover data. Since the methodology is applicable to many cell types and modifications, it has substantial potential to prioritize PTMs for functional investigation in the future.
Project description:To understand the extent that Heat shock protein 90 (Hsp90) regulated its target proteins at the transcription level, transcriptomic change was profiled in yeast cells upon Hsp90 compromising. We genetically modified the R1158 strain (resulting genotype of mutant strain: TETp-HSC82 hsp82Δ arg4Δ lys5Δ car2Δ::URA3) and then reduced the Hsp90 amount with doxycycline treatment. Fold change of mRNA from untreated to treated cells indicated the transcriptomic change. Totally, we identified 1104 genes mis-regulated with a fold change of no less than 1.5 (P <0.05) upon Hsp90 compromising. Two-condition experiment, treated vs. untreated cells. Biological duplicates, independently grown and harvested. Technical triplicates for RNA isolation.
Project description:Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study transcriptomic and proteomic changes in the estrogen receptor negative, non tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R=0.679, P<0.05) and KEAP1 knockdown (R=0.853, P<0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and proteomic level. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast. MCF10A cells were treated with SFN or had KEAP1 knocked down by siRNA.
Project description:The immunopeptidome is constantly monitored by T cells to detect foreign or aberrant HLA peptides. It is highly dynamic and reflects the current cellular state, enabling the immune system to recognize abnormal cellular conditions, such as those present in cancer cells. To precisely determine how changes in cellular processes, such as those induced by drug treatment, affect the immunopeptidome, quantitative immunopeptidomics approaches are essential. To meet this need, we developed a pulsed SILAC-based method for quantitative immunopeptidomics. Metabolic labeling with lysine, arginine, and leucine enabled isotopic labeling of nearly all HLA peptides across all HLA allotypes (> 90% on average). We established a data analysis workflow that integrates the de novo sequencing-based tool Peptide-PRISM for comprehensive HLA peptide identification with MaxQuant for accurate quantification. We employed this strategy to explore the modulation of the immunopeptidome upon MAPK pathway inhibition and to investigate alterations associated with acquired resistance to BRAF and MEK inhibitors. Our analyses demonstrated significant changes in the immunopeptidome following MAPK pathway inhibition, as well as in cells resistant to BRAF/MEK inhibitors. Moreover, we identified putative tumor-specific cryptic HLA peptides linked to these processes.
Project description:Protein expression is regulated by production and degradation of mRNAs and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics to build a quantitative genomic model of the differential regulation of gene expression in LPS stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for over half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction of novel cellular functions and remodeling of preexisting functions through the protein life cycle. Mouse primary dendritic cells were treated with LPS or mock stimulus and profiled over a 12-hour time course. Cells were grown in M-labeled SILAC media, which was replaced with H-labeled SILAC media at time 0. Aliquots were taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 9, and 12 hours post-stimulation and added to equal volumes of a master mix of unlabeled (L) cells for the purpose of normalization. RNA-Seq was performed at 0, 1, 2, 4, 6, 9, and 12 hours post-stimulation.