Project description:Mitofusin-1 (MFN1) and Mitofusin-2 (MFN2) are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria yuxtaposition, and autophagy. However, the mechanisms by which these proteins participate in these processes remain poorly understood. To better understand their functions, we studied the interactomes of these two proteins. To this end, we used CRISPR/Cas9 technology to insert an HA-tag in the C-terminal domain of MFN1 and MFN2 and generated HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA, respectively. HA-pulldown followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorganelle communication. We characterized RAB5C as an endosomal modulator of mitochondrial dynamics through its interaction with MFN1 and SLC27A2 as a novel partner of MFN2 relevant in autophagy. Our findings reveal that MFN proteins participate in nutrient-modulated pathways involved in organelle communication.
Project description:A series of valdiazen chemical probes were developed and applied in affinity-based pulldown experiments to enrich potential valdiazen-binding proteins.
Project description:Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3 UB ligases remains a challenge. We overcame this challenge by constructing an “orthogonal UB transfer (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2 that are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.
Project description:The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity, and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. The Estrogen Receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Applying interaction proteomics coupled to mass spectrometry (MS) to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo both in the nucleus and in cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association to a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ+ vs ERβ- cells, and before and after AGO2 knock-down in ERβ+ cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may able to control directly also mRNA translation efficiency and stability.These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions
Project description:To examine the protein composition of germ granules subcompartments of C. elegans, while ensuring their physiological integrity, we utilized TurboID-based proximity biotin labeling techniques to idedtify the protein components of P granules, Z granules and Mutator foci in the germline of C.elegans.
Project description:Cerebral organoids â three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells â have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures. 734 single-cell transcriptomes from human fetal neocortex or human cerebral organoids from multiple time points were analyzed in this study. All single cell samples were processed on the microfluidic Fluidigm C1 platform and contain 92 external RNA spike-ins. Fetal neocortex data were generated at 12 weeks post conception (chip 1: 81 cells; chip 2: 83 cells) and 13 weeks post conception (62 cells). Cerebral organoid data were generated from dissociated whole organoids derived from induced pluripotent stem cell line 409B2 (iPSC 409B2) at 33 days (40 cells), 35 days (68 cells), 37 days (71 cells), 41 days (74 cells), and 65 days (80 cells) after the start of embryoid body culture. Cerebral organoid data were also generated from microdissected cortical-like regions from H9 embryonic stem cell derived organoids at 53 days (region 1, 48 cells; region 2, 48 cells) or from iPSC 409B2 organoids at 58 days (region 3, 43 cells; region 4, 36 cells).
Project description:The RNA-binding protein Trim71/Lin41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development and cancer. Trim71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Deposited data are mass spectrometry data from immunoprecipitation experiments with endogenously tagged Tnrc6a. Experiments are carried out in mouse embryonic stem cells (mESCs) in the presence and absence of RNase A.
Project description:The RNA-binding protein Trim71/Lin41 is a phylogenetically conserved developmental regulator that functions in mammalian stem cell reprogramming, brain development and cancer. Trim71 recognizes target mRNAs through hairpin motifs and silences them through molecular mechanisms that await identification. Deposited data are mass spectrometry data from immunoprecipitation experiments with endogenously tagged Trim71, Ago2, and Tnrc6a. Experiments are carried out in mouse embryonic stem cells (mESCs) and uncover reciprocal interactions of Trim71, Ago2, and Tnrc6a. Trim71 protein interactions are largely independent of Ago2 levels, but strongly depend on the presence of RNA. A second set of experiments is mass spectrometry data from immunoprecipitation experiments in mESCs overexpressing a tagged peptide derived from Homo sapiens TNRC6B (‘FLAG-HA::T6BWT::Cherry’), that is known to block the Tnrc6-Ago2 interaction. We show that a wild-type version strongly binds Ago1 and Ago2, but not Trim71, while a mutant version binds neither Ago1, 2, nor Trim71.
Project description:The canonical NF-κB pathway is active in 70% of all pancreatic cancer cases and NF-κB Essential Modulator (NEMO) is essential for the activation of this pathway. In our study, we used KC mice, which express the oncogenic KRAS and develop precancerous lesions termed Pancreatic Intraepithelial Neoplasias (PanINs), and KNeC mice, which express the oncogenic KRAS and have NEMO deleted in their pancreatic cells. These mice were injected with cerulein to promote the development of pancreatitis (cerulein dosage= 50μg/kg). Cerulein was injected at 8 hourly intervals for 2 days in total. The first injection day was when mice reached their sixth week of age and the second injection day was 3 days after the first injection day. Both KC and KNeC mice developed PanINs. At the age of 10 months, pancreata of KC and KNeC mice were analyzed. Using laser capture microdissection, PanINs from both groups were excised and their transcriptome was analyzed though RNA-seq.
Project description:Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting synovial joints and leading to cartilage damage and bone loss. This destruction is promoted by activated fibroblast-like synoviocytes (FLS) that show an invasive and migratory phenotype. The mechanisms of FLS activation are unknown, but evidence suggests that pre-damaged extracellular matrix (ECM) of the cartilage can trigger FLS activation. Integrin α11β1 might be involved in the activation, as it is highly increased in the synovium of RA patients and hTNFtg mice, an RA mouse model. Since TNFα is the major cytokine induced in RA, we treated murine chondrocytes with TNFα to produce a damaged, RA-like matrix. Comparison to healthy chondrocyte matrix revealed decreased ECM proteins, including several collagens and proteoglycans, increased matrix-degrading proteins and elevated levels of inflammatory cytokines. FLS responded to those differences in the damaged chondrocyte matrix with a matrix-remodeling and pro-inflammatory phenotype characterized by expressing genes involved in matrix degradation and increased production of CLL11 and CCL19. Damaged chondrocyte matrix induced increased Itga11 expression in FLS, which correlates with the increased α11β1 amounts in RA patients. FLS deficient in integrin α11β1 released lower amounts of inflammation-associated cytokines but did not reveal significant differences from the response of wild type FLS to a damaged, RA-like matrix. Our results demonstrate differences in healthy and RA-like chondrocyte ECM and distinctly different responses of wt FLS to damaged versus healthy ECM.