Project description:To identify the specific proteins interacting with STX17, HEK293T cells stably expressing Flag-STX17 were lysed and immunoprecipitated with anti-Flag antibody, and eluted for Commassie blue staining and then subjected to mass spectrometric analysis.
Project description:Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a cytosolic protein kinase that regulates multiple inflammatory and cell death pathways. Serine/Threonine phosphorylation of RIPK1 is known to suppress RIPK1 kinase-mediated cell death in the contexts of inflammation, infection and embryogenesis, however, regulation by tyrosine phosphorylation has not been reported. Here, we show that non-receptor tyrosine kinases Janus kinase 1 (JAK1) and SRC are able to phosphorylate RIPK1 at Y384 (Y383 in murine RIPK1), leading to suppression of TNF-induced cell death. Mice bearing a homozygous Ripk1 mutation that prevents tyrosine phosphorylation of RIPK1 (Ripk1Y383F/Y383F), develop systemic inflammation and emergency haematopoiesis. Mechanistically, Ripk1Y383F/Y383F mutation promotes RIPK1 kinase activation and enhances TNF-induced apoptosis and necroptosis, which is partially due to impaired recruitment and activation of MAP kinase-activated protein kinase 2 (MK2). The systemic inflammation and emergency haematopoiesis in Ripk1Y383F/Y383F mice are largely alleviated by RIPK1 kinase inhibition, and prevented by genomic deletions targeted to the upstream pathway (either to Tumor necrosis factor receptor 1 or RIPK3 and Caspase8 simultaneously). In summary, our results demonstrate that tyrosine phosphorylation of RIPK1 is critical for regulating RIPK1 activity to limit cell death and inflammation.
Project description:In order to investigate the dynamic regulation of the APC/C by intrinsically disordered regions (IDRs) upon phosphorylation, we employed cross-linking mass spectrometry (CLMS). Given that the phosphorylation of Apc1-300L (IDR in Apc1) is a key determinant in its release from the co-activator binding site, we compared the interaction profiles of unphosphorylated and hyper-phosphorylated rAPC/C_WT complexes. The CLMS analysis highlights not only known interactions consistently identified within APC/C complexes, regardless of phosphorylation status, but also dynamic IDR-mediated interactions that are lost upon phosphorylation or are phosphorylation-dependent. Additionally, the data indicate that phosphorylation significantly reduces Apc1-300L's interaction with Apc8-L (IDR in Apc8) and Apc6, suggesting Apc1-300L's dislocation from the co-activator Cdc20 binding site, thereby facilitating Cdc20 loading onto the APC/C.
Project description:The Tau (MAPT) protein drives neuronal dysfunction and toxicity in the brain in Alzheimer’s disease (AD) and other Tauopathies. To dissect the complexity of the Tau interactome that underlies this process we used two proteomic approaches to characterize the dynamic and multifaceted nature of Tau protein-protein interactions in human induced pluripotent stem cell (iPSC)-derived neurons. We used engineered ascorbic acid peroxidase (APEX) for spatiotemporally restricted mapping of Tau interaction proteins in combination with quantitative affinity purification mass spectrometry (AP-MS) to interrogate disease-related changes in the Tau interactome. The APEX method resolved subcellular interactions of wild-type Tau at amino acid level resolution in living neurons as well as novel activity-dependent interactions of Tau with presynaptic vesicle proteins that occurred during Tau secretion from neurons. Among the many Tau interacting proteins revealed by AP-MS, the interaction of mitochondrial proteins with wild-type Tau (TauWT) was more robust than with TauV337M or TauP301L. The mitochondrial proteins that preferentially interacted with TauWT comprised a protein module that is downregulated in multi-omics analyses of human AD brains. Mitochondrial bioenergetics were altered in TauV337M compared to TauWT human iPSC-derived neurons confirming the impact of Tau on mitochondria. These Tau interactome analyses open up novel disease-related processes as potential therapeutic targets to block Tau-mediated pathogenesis.
Project description:Influenza B virus (IBV) strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens' eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for IBVs, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 IBV strains from 1940 to present day, representing the known temporal and genetic variability of IBV circulating in humans. This panel contains strains from the B/Victoria/2/87-like lineage, B/Yamagata/16/88-like lineage and the ancestral lineage that preceded their split to provide a diverse set that would help to identify a suitable backbone which can be used in combination with hemagglutinin (HA) and neuraminidase (NA) glycoproteins from any IBV strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 IBV strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to select for high-growth reassortant viruses. After serial passaging, we selected 10 clonal isolates based on their growth profiles assessed by hemagglutination and plaque-forming units. We then generated reverse genetics systems for the three clones that performed best in growth curves. The selected backbones were then used to generate different reassortant viruses with HA/NA combinations from high and low titer yielding wild type IBV. When the growth profiles of the recombinant reassortant viruses were tested, the low titer yielding HA/NA viruses with the selected backbones yielded higher titers similar to those from high titer yielding HA/NA combinations. The use of these IBV backbones with improved replication in eggs might increase yields for the influenza B virus components of seasonal influenza virus vaccines.
Project description:TGFBIp-related corneal dystrophy (CD) is the most common form of stromal corneal dystrophy. Although CD is a group of inheritable and progressive corneal diseases, non-inheritable cases of corneal amyloid deposition are reported. TGFBIp-CD is characterized by the accumulation of insoluble protein deposits consisting smaller proteolytic fragments of TGFBIp in the corneal tissues, eventually leading to progressive corneal opacity. Maximum density of these aggregates is in the corneal center implicating the local absence of amyloid controlling factors. Currently, no other treatment options are available besides the surgical replacement of the affected corneal tissues with a donor’s cornea. Here we show that neuroprotective ATP-independent amyloid β chaperone L-PGDS abundant in cerebrospinal fluid but absent in the corneal tissues can effectively disaggregate amyloids in vitro and in the surgically excised human cornea of patients with TGFBIp-CD.
Project description:In order to study the molecular mechanism of FBL in cells, this project constructed the full length of FBL into the SFB (S-Flag-SBP tag) vector, and constructed a stable cell line overexpressing FBL. Affinity chromatography combined with LC-MS/MS technology to screen FBL interacting proteins.
Project description:NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals, but few of their substrates are known. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity via spatial regulation of Gef1, a guanine nucleotide exchange factor for the small GTPase Cdc42. Here we show that Orb6 plays a critical role as a positive regulator of exocytosis, independent of Gef1. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify several proteins involved in membrane trafficking as Orb6 targets, and we confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro. Our results suggest that Orb6 kinase activity is crucial for exocyst localization to actively-growing cell tips and for exocyst activity during septum dissolution after cytokinesis. We further show that Orb6 phosphorylation of Sec3 serine-201 contributes to exocyst function in parallel with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels.