ABSTRACT: Characterization of the proteome of extracellular vesicles produced by human neuroblastoma cells undergoing karyoptosis by autophagy inhibition
Project description:Proteins co-localizing with RNA targets of interest in human induced pluripotent stem cells (iPSCs) were identified using our recently optimized hybridization-proximity labeling approach (HyPro2) combined with label-free mass spectrometry. Briefly, fixed and permeabilized iPSCs from an ALS patient (DN19V4) or a healthy donor (C0053) were hybridized with a digoxigenin-labeled antisense oligonucleotide probe targeting transcripts containing the G4C2 hexanucleotide repeat expansion, which is present in the C9orf72 gene in DN19V4 but not in C0053. Alternatively, a control probe set was used to target ACTB pre-mRNA, expressed in both DN19V4 and C0053. The purified HyPro2 enzyme, which contains a digoxigenin-binding domain and a modified ascorbate peroxidase domain, was then recruited to the RNA targets, enabling in vitro biotinylation of target-proximal proteins. Following streptavidin pull-down, biotinylated proteins were fragmented using a Trypsin/Lys-C protease mix and identified by label-free LC-MS/MS analysis. All experiments were performed in triplicate
Project description:Proteins co-localizing with RNA targets of interest in HeLa cells were identified using our recently optimized hybridization-proximity labeling approach (HyPro2) combined with label-free mass spectrometry. Briefly, fixed and permeabilized cells were hybridized with digoxigenin-labeled antisense oligonucleotide probes targeting the long noncoding RNA PNCTR or pre-mRNAs encoding ACTB and FUS proteins. A no-probe negative control was also included. The purified HyPro2 enzyme, which contains a digoxigenin-binding domain and a modified ascorbate peroxidase domain, was then recruited to the RNA targets, enabling in vitro biotinylation of target-proximal proteins. Following streptavidin pull-down, biotinylated proteins were fragmented using a Trypsin/Lys-C protease mix and identified by label-free LC-MS/MS analysis. All experiments were performed in triplicate.
Project description:TDP-43 is a primary pathogenic protein in both amyotrophic lateral sclerosis and frontotemporal dementia, but it’s accumulation and aggregation show regional variation, with abundant deposition in the cortex, and a much lower burden in the cerebellum. Here we use mass spectrometry analysis to compare the protein interactome of TDP-43 within the cortex and cerebellum of young healthy C57Bl/6J mice.
Project description:NGN3 is a transcription factor whose transient expression during pancreatic development is vital for the generation of endocrine pancreatic cells, including beta cells. NGN3 stabilisation has been shown to induce exocrine-to-endocrine cell plasticity in the murine pancreas, making it a viable target for therapies aiming to replenish beta cells after immune-mediated destruction in type 1 diabetes patients. Here, we set out to identify new interactors of NGN3 that could play a role in its post-translational regulation. We transfected HEK293A cells with HA-tagged NGN3 and carried out immunoprecipitation of the HA-tag, followed by analysis of co-immunoprecipitated interactors via LC-MS/MS.
Project description:Productive infection by human immunodeficiency virus type-1 (HIV-1) requires the import of viral replication complexes into the nuclei of infected cells. Myxovirus resistance 2 (MX2/MxB) blocks this step, halting nuclear accumulation of viral DNA and virus replication. Here, we identify positions in MX2 whose phosphorylation status reduces or enhances antiviral function (hypomorphic and hypermorphic variants, respectively). Importantly, hypermorphic mutant proteins not only increased inhibitory activity against wild-type HIV-1 but can also exhibit antiviral capabilities against HIV-1 capsid mutant viruses that are resistant to wild-type MX2. Furthermore, some of these proteins were also able to inhibit retroviruses that are insensi- tive to MX2. Therefore, we propose that phosphorylation comprises a major element of MX2 regulation and substrate determination.
Project description:To identify the cellular localisation of the HpdBCA decarboxylase complex, a plasmid based HpdB-SNAP-tag translational fusion was constructed using a C. difficile compatible plasmid (pMTL84151) under control of the hpdBCA promoter. The hpdB coding sequence omitting the stop codon, was fused via a linker to a SNAP-tag (Table 1). Confirmation of HpdB linked to the SNAP-tag was carried out via western blot, and mass spectrometry, in which we identified six peptides, four with 100% identity and two with 99% identity unique to HpdB. Localisation of HpdB was visualised by confocal microscopy in 630Δerm and the p-cresol deficient mutant (hpdC::CT) (carrying the HpdB-SNAP-tag fusion (PhpdB-CDS-SNAP), in the presence and absence of p-HPA) to induce HpdBCA production.
Project description:Oxidants induce cell cycle arrest to halt cell proliferation; however, little is known about the redox-regulated effector proteins that mediate these processes. Here, we report a novel kinase-inhibitory disulfide bond in cyclin D-CDK4 and investigate its role in cell proliferation and PH. Oxidative modifications of cyclin D-CDK4 were detected in human pulmonary arterial smooth muscle cells (HPASMCs) and human pulmonary arterial endothelial cells (HPAECs). Cysteine to alanine mutants were generated, and cell cycle experiments were employed to characterize the nature of this reversible intermolecular disulfide bond. The functional role of the disulfide was delineated using in vitro kinase activity assays, HPASMCs and knock-in cells. Finally, the cyclin D-CDK4 disulfide was assessed in vivo in the pulmonary arteries and isolated HPASMCs of PAH patients, and in three preclinical models of PH. Cyclin D-CDK4 forms an oxidant-induced heterodimeric disulfide dimer between C7/8 and C135, respectively. This reversible modification forms in cells in vitro and in pulmonary arteries in vivo to inhibit cyclin D-CDK4 kinase activity and decrease retinoblastoma protein (Rb) phosphorylation. Correspondingly, treatment of HPASMCs with H2O2 or auranofin induces cell cycle arrest. Notably, mutation of CDK4 C135 causes a kinase-impaired phenotype, which decreases the proliferation rate of cells, suggesting this cysteine is indispensable for cyclin D-CDK4 kinase activity. Pulmonary arteries and HPASMCs from patients with pulmonary arterial hypertension (PAH) display a decreased level of CDK4 disulfide, consistent with CDK4 being hyperactive in PAH. Furthermore, auranofin treatment, which induces the cyclin D-CDK4 disulfide, attenuates disease severity in experimental models of PH, by mitigating pulmonary vascular remodeling. A novel disulfide bond in cyclin D-CDK4 acts as a rapid switch to inhibit kinase activity and halt cell proliferation. This oxidative modification forms at a critical cysteine residue, which is unique to CDK4, offering the potential for design of a selective covalent inhibitor that may prove beneficial in pulmonary hypertension
Project description:Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular ageing. Sox9 (SRY-Box Transcription Factor 9) is a master regulator of chondrogenesis, also expressed in the vasculature, that has been implicated in vascular smooth muscle cell (VSMC) osteo-chondrogenic conversion. Here, we investigated the relationship between vascular ageing, calcification and Sox9-driven ECM regulation in VSMCs. Immunohistochemistry in human aortic samples showed that Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16. Analysis of Sox9 expression in vitro showed it was mechanosensitive with increased expression and nuclear translocation in senescent cells and on stiff matrices. Manipulation of Sox9 via overexpression and depletion, combined with atomic force microscopy (AFM) and proteomics, revealed that Sox9 regulates ECM stiffness and organisation by orchestrating changes in collagen expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs whereby senescent cells plated onto ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (LH3) was identified as a Sox9 target, and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles (EVs) and Sox9 promoted EV secretion, leading to increased LH3 deposition within the ECM. These findings identify cellular senescence and Sox9 as a key regulators of ECM stiffness during VSMC ageing and highlight a crucial role for ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle whereby cellular senescence and increased ECM stiffening promote Sox9 expression which drives further ECM modifications that act to accelerate vascular stiffening and cellular senescence.
Project description:Neural crest cells are multipotent cells that delaminate from the neuroepithelium, migrating throughout the embryo. Aberrant migration causes developmental defects. Animal models are improving our understanding of neural crest anomalies, but in vivo migration behaviours are poorly understood. Here, we demonstrate that murine neural crest cells display actin-based lamellipodia and filopodia in vivo. Using neural crest-specific knockouts or inhibitors, we show that the serine-threonine kinase Glycogen Synthase Kinase-3 (GSK3), and the cytoskeletal regulator Lamellipodin (Lpd), are required for lamellipodia formation whilst preventing focal adhesion maturation. Lpd is a novel substrate of GSK3 and phosphorylation of Lpd favours interactions with the Scar/WAVE complex (lamellipodia formation) at the expense of VASP and Mena interactions (adhesion maturation and filopodia formation). This improved understanding of cytoskeletal regulation in mammalian neural crest migration has general implications for neural crest anomalies and cancer.
Project description:Tauopathies are characterized by the progressive accumulation of abnormal tau species, which disrupt the autophagy-lysosomal pathway (ALP), a critical system for degrading intracellular macromolecules and aggregated proteins, causing toxicity and cell death. This study investigates the impact of the N-terminally truncated Tau35 protein overexpression on proteolytic pathways, including effects on autophagy and endo-lysosomal processes. Using a Tau35 mouse model and SH-SY5Y cell lines stably expressing either the Tau35 fragment or full-length tau, we employed western blotting, proteomic analysis of lysosome-enriched brain fractions, proteolysis/endocytosis assays, and live-cell imaging with the lysotracker reporter to assess protein degradation and lysosomal function. Our findings identify early pathological changes in endo-lysosomal processes, including increased endocytosis, proteolytic dysfunction and lysosomal motility abnormalities, associated with Tau35- induced toxicity.