The nucleolus is a distinct subnuclear compartment that was first observed more than 200 years ago. Nucleoli assemble around the tandemly repeated ribosomal DNA gene clusters and 28S, 18S and 5.8S ...ribosomal RNAs (rRNAs) are transcribed as a single precursor, which is processed and assembled with the 5S rRNA into ribosome subunits. Although the nucleolus is primarily associated with ribosome biogenesis, several lines of evidence now show that it has additional functions. Some of these functions, such as regulation of mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles, will be discussed, as will the relation of the nucleolus to human diseases.
Glycogen synthase kinase-3 (GSK3) are ubiquitously expressed serine-threonine kinases involved in a plethora of functions ranging from the control of glycogen metabolism to transcriptional ...regulation. We recently demonstrated that GSK3 inhibition triggers JNK-cJUN-dependent apoptosis in human pancreatic cancer cells. However, the comprehensive picture of downstream GSK3-regulated pathways/functions remains elusive. Herein, counterbalancing the death signals, we show that GSK3 inhibition induces prosurvival signals through increased activity of the autophagy/lysosomal network. Our data also reveal a contribution of GSK3 in the regulation of the master transcriptional regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB) in pancreatic cancer cells. Similarly to mammalian target of rapamycin (mTOR) inhibition, GSK3 inhibitors promote TFEB nuclear localization and leads to TFEB dephosphorylation through endogenous serine/threonine phosphatase action. However, GSK3 and mTOR inhibition impinge differently and independently on TFEB phosphorylation suggesting that TFEB is regulated by a panel of kinases and/or phosphatases. Despite their differential impact on TFEB phosphorylation, both GSK3 and mTOR inhibitors promote 14-3-3 dissociation and TFEB nuclear localization. Quantitative mass spectrometry analyses further reveal an increased association of TFEB with nuclear proteins upon GSK3 and mTOR inhibition suggesting a positive impact on TFEB transcriptional function. Finally, a predominant nuclear localization of TFEB is unveiled in fully fed pancreatic cancer cells, whereas a reduction in TFEB expression significantly impairs their capacity for growth in an anchorage-independent manner. In addition, TFEB-restricted cells are more sensitive to apoptosis upon GSK3 inhibition. Altogether, our data uncover new functions under the control of GSK3 in pancreatic cancer cells in addition to providing key insight into TFEB regulation.
Glycogen synthase kinase-3 (GSK3) inhibition promotes apoptosis of pancreatic cancer cells.
GSK3 inhibition concomitantly triggers a prosurvival autophagic response.
Autophagy inhibition promotes GSK3 inhibition-induced apoptosis.
GSK3 has a dual role in pancreatic cancer cell survival.
Cells typically respond quickly to stress, altering their metabolism to compensate. In mammalian cells, stress signaling usually leads to either cell-cycle arrest or apoptosis, depending on the ...severity of the insult and the ability of the cell to recover. Stress also often leads to reorganization of nuclear architecture, reflecting the simultaneous inhibition of major nuclear pathways (e.g., replication and transcription) and activation of specific stress responses (e.g., DNA repair). In this review, we focus on how two nuclear organelles, the nucleolus and the Cajal body, respond to stress. The nucleolus senses stress and is a central hub for coordinating the stress response. We review nucleolar function in the stress-induced regulation of p53 and the specific changes in nucleolar morphology and composition that occur upon stress. Crosstalk between nucleoli and CBs is also discussed in the context of stress responses.
The elucidation of the subcellular distribution of proteins under different conditions is a major challenge in cell biology. This challenge is further complicated by the multicompartmental and ...dynamic nature of protein localization. To address this issue, quantitative proteomics workflows have been developed to reliably identify the protein complement of whole organelles, as well as for protein assignment to subcellular location and relative protein quantification based on different cell culture conditions. Here, we review quantitative MS‐based approaches that combine cellular fractionation with proteomic analysis. The application of these methods to the characterization of organellar composition and to the determination of the dynamic nature of protein complexes is improving our understanding of protein functions and dynamics.
The elucidation of the subcellular distribution of proteins under different conditions is a major challenge in cell biology. This challenge is further complicated by the multicompartmental and dynamic nature of protein localization. Here, we review quantitative mass spectrometry‐based approaches which combine cellular fractionation with proteomic analysis.
Phenotypic Analysis of Organoids by Proteomics Gonneaud, Alexis; Asselin, Claude; Boudreau, François ...
Proteomics (Weinheim),
October 2017, 2017-Oct, 2017-10-00, 20171001, Volume:
17, Issue:
20
Journal Article
Peer reviewed
The development of 3D cell cultures into self‐organizing organ‐like structures named organoids provides a model that better reflects in vivo organ physiology and their functional properties. ...Organoids have been established from several organs, such as the intestine, prostate, brain, liver, kidney and pancreas. With recent advances in high‐throughput and ‐omics profiling technologies, it is now possible to study the mechanisms of cellular organisation at the systems level. It is therefore not surprising that these methods are now used to characterize organoids at the transcriptomic, proteomic, chromatin state and transcription factor DNA‐binding levels. These approaches can therefore provide a wealth of information regarding both the mechanisms involved in different diseases, and those involved in cell responses to different conditions, in a more in vivo setting. The authors provide an overview of the potential applications of quantitative mass spectrometry with organoid culture, and how the use of large‐scale proteome measurements is emerging in different organoid systems.
Objective
Mitochondria are organelles that exhibit several bacterial features, such as a double‐stranded genome with hypomethylated CpG islands, formylated proteins, and cardiolipin‐containing ...membranes. In systemic lupus erythematosus (SLE), mitochondria and their inner components are released into the extracellular space, potentially eliciting a proinflammatory response from the immune system. While cardiolipin and mitochondrial DNA and RNA are confirmed targets of autoantibodies, other antigenic mitochondrial proteins in SLE remain to be identified. The present study was undertaken to characterize the protein repertoire recognized by antimitochondrial antibodies (AMAs) in patients with SLE.
Methods
Using shotgun proteomic profiling, we identified 1,345 proteins, 431 of which were associated with the mitochondrial proteome. Immunoreactivities to several of these candidate proteins were assessed in serum samples from a local cohort (n = 30 healthy donors and 87 patients with SLE) using enzyme‐linked immunosorbent assay, and further analyzed for associations with demographic and disease characteristics.
Results
We determined that IgG antibodies to the complement component C1q binding protein were significantly elevated in the patients with SLE (P = 0.049) and were also associated with lupus anticoagulant positivity (P = 0.049). Elevated levels of IgG antibodies against mitochondrial protein mitofusin 1 (MFN‐1) were promising predictors of SLE diagnosis in our cohort (adjusted odds ratio 2.99 95% confidence interval 1.39–6.43, P = 0.0044). Moreover, increased levels of anti–MFN‐1 were associated with the presence of antiphospholipids (P = 0.011) and anti–double‐stranded DNA (P = 0.0005).
Conclusion
In this study, we characterized the mitochondrial repertoire targeted by AMAs in the setting of SLE. Our results indicate that autoantibodies can recognize secreted and/or surface proteins of mitochondrial origin. Profiling of the AMA repertoire in large prospective cohorts may improve our knowledge of mitochondrial biomarkers and their usefulness for patient stratification.
The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that ...combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.
Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome ...era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ∼20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.
Understanding the complex network of protein-protein interactions (PPI) that govern cellular functions is essential for unraveling the molecular basis of biological processes and diseases. Mass ...spectrometry (MS) has emerged as a powerful tool for studying protein dynamics, enabling comprehensive analysis of protein function, structure, post-translational modifications, interactions, and localization. This article provides an overview of MS techniques and their applications in proteomics studies, with a focus on the replication fork proteome. The replication fork is a multi-protein assembly involved in DNA replication, and its proper functioning is crucial for maintaining genomic integrity. By combining quantitative MS labeling techniques with various data acquisition methods, researchers have made significant strides in elucidating the complex processes and molecular mechanisms at the replication fork. Overall, MS has revolutionized our understanding of protein dynamics, offering valuable insights into cellular processes and potential targets for therapeutic interventions.
RAB GTPases are central modulators of membrane trafficking. They are under the dynamic regulation of activating guanine exchange factors (GEFs) and inactivating GTPase‐activating proteins (GAPs). ...Once activated, RABs recruit a large spectrum of effectors to control trafficking functions of eukaryotic cells. Multiple proteomic studies, using pull‐down or yeast two‐hybrid approaches, have identified a number of RAB interactors. However, due to the in vitro nature of these approaches and inherent limitations of each technique, a comprehensive definition of RAB interactors is still lacking. By comparing quantitative affinity purifications of GFP:RAB21 with APEX2‐mediated proximity labeling of RAB4a, RAB5a, RAB7a, and RAB21, we find that APEX2 proximity labeling allows for the comprehensive identification of RAB regulators and interactors. Importantly, through biochemical and genetic approaches, we establish a novel link between RAB21 and the WASH and retromer complexes, with functional consequences on cargo sorting. Hence, APEX2‐mediated proximity labeling of RAB neighboring proteins represents a new and efficient tool to define RAB functions.
Synopsis
Quantitative affinity purification combined with mass spectrometry (AP‐MS) and APEX2‐mediated proximity labeling of early endosomal RAB GTPases identify a novel interaction between RAB21 and the WASH/retromer complexes. Functional validation of the proteomic data defines a role for RAB21 in endosomal sorting of a subset of clathrin‐independent cargos.
Unbiased proteomics establish a network of early endosomal RAB interacting and neighboring proteins.
RAB21 interacts with the WASH and retromer complexes, and acts as a modulator of F‐actin generation at endosomes.
RAB21 regulates sorting of a subset of clathrin‐independent cargos, such as MCT1, SLC3A2, Basigin and CD44.
Quantitative proteomics and APEX2‐mediated proximity labeling of early endosomal RAB GTPases identify a novel interaction between RAB21 and the WASH/retromer complexes and defines a role for RAB21 in endosomal sorting of a subset of clathrin‐independent cargos.
PDF
