Project description:The 293T cells overexpressing human telomerase reverse transcriptase (hTERT) were lysed and co-immunoprecipitation was performed using hTERT antibody. Then protein mass spectrum was conducted in order to identify the hTERT-interacting proteins.
Project description:Subversion of the host cytoskeleton is a critical virulence mechanism used by a variety of intracellular bacterial pathogens during their infectious life cycles. Growing evidence suggests that the tactics employed by pathogens are surprisingly diverse. Using proteomic and biochemical methods, we demonstrate that the S. Tm type III effector protein SopB potently interacts specifically with host cell IFs vimentin. After host cell entry, Salmonella replicates in Salmonella containing vacuoles (SCVs), which accumulate a dense meshwork of vimentin through the Salmonella SPI-1 type III secretion effector SopB. We also demonstrate an important role for SopB-vimentin interaction, in the cellular responses including pro-inflammatory cytokine production induced by Salmonella. Vimentin interacts with the N-terminus of the S. Tm T3SS effector protein SopB. Vimentin and its interaction with SopB are dispensable for S. Tm invasion, but are required for stable formation of SCVs which are essential for bacteria replication. Moreover, SopB lacking of N-terminus cdc42-binding domain loses the interaction with vimentin, suggesting that the small GTPase is critical in SopB triggered vimentin recruitment to form integrative SCVs. These findings establish that SCVs integrity of the bacterium specifically requires intermediate filaments vimentin, which is a prerequisite for highly efficient S. Tm replication.
Project description:: Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET & HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18, we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3-II and p62 levels, indicating increased autophagosome formation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (IPSCs). Here, loss of VPS18 resulted in an almost complete absence of developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring IPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.
Project description:Streptococcus suis is a zoonotic pathogen with a high incidence and mortality rate in both swine and humans. Following antibiotic treatment, the organism has evolved many resistance mechanisms, among of which efflux pump overexpression can promote drug extrusion out of the cell. This study clarified the role of CiaRH in fluoroquinolone resistance. A deletion of the ciaRH genes showed decreased susceptibility to tested antibiotics, an invariant growth rate, and reduced intracellular substrates. This research also demonstrated that the overexpression of efflux pump, SatAB, was the main cause of ∆ciaRH resistance. In addition, CiaR could combine with the promoter region of satAB, to further directly suppress target gene transcription. Simultaneously, satAB was also directly regulated by SatR. Our findings may provide novel insight into the development of drug targets, and help exploit corresponding inhibitors to combat bacterial multidrug resistance.
Project description:The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. While this structure is known to be important for fertilization, little is known regarding its proteomic composition. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus. To explore the proteomic composition of the PT we performed the first in depth, label-free proteomic characterization of the PT of boar spermatozoa. To do this, a unique subcellular fractionation protocol was first performed to isolate the PT and increase our ability to detect lowly abundant sperm proteins. Through the use of this subcellular fractionation technique we were able to quantify 1802 proteins, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire known proteome of the Sus scrofa species so far. In the PT structure itself we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data also revealed that the PT is replete in proteins critical for sperm-egg fusion including: Izumo sperm-egg fusion proteins 1-4 (IZUMO1-4) and phosphoinositide phospholipase C (PLCZ1).
Project description:In this study, proteomic analysis on ZIKV-infected primary human fetal neural progenitor cells (NPCs) revealed that virus infection altered levels of cellular proteins involved in NPC proliferation, differentiation and migration.
Project description:Spliceosome dephosphorylation is essential control step that enables intron removal from pre-mRNA for the regulation of gene expression. However, the phosphatase that is biologically responsible has not been identified. Here we show that PP2A B́ η, a B subunit of PP2A phosphatase, associates with spliceosome B* to C* complex and dephosphorylates spliceosome regulators by the most-conserved its binding motif that can be absolutely regimented interaction with PP2A substrates.
Project description:Cells sense and adapt to their environment. Parasitic protists for example, such as African trypanosomes, occupy distinct environments in mammalian hosts, and in insect vectors, and adapt energy metabolism and growth accordingly, but the mechanisms involved remain incompletely characterized. Here we describe environmental sensing dependent upon Trypanosoma brucei Quick IQ-motif protein 1 (QIQ1). We use a genome-scale loss-of-function genetic screen to identify knockdowns associated with gain-of-fitness, or a ‘quick’ growth phenotype, in bloodstream-form cells. The dominant hit, QIQ1 (Tb927.8.6870), encodes a flagellum-localized protein with multiple putative calmodulin-binding IQ-motifs. In a competition assay, qiq1-null cells displayed a density-dependent growth advantage, which was abrogated by calcium chelation. When cells were grown at reduced density in blood serum, with a series of supplements, only bicarbonate restored the qiq1-null competitive advantage. Proteomic analysis indicated that QIQ1 increased the expression of carnitine acetyltransferase, suggesting a role in modulating fatty acid metabolism, while expression of a flagellum-localized calmodulin was both QIQ1 and bicarbonate-dependent. We conclude that trypanosomes sense and adapt to distinct environments in a QIQ1-dependent manner. Our findings suggest that QIQ1-signaling facilitates CO2-sensing to optimize utilization of available nutrients via a pathway involving calcium, calmodulin, and carnitine.
Project description:Signal-sequence-lacking superoxide dismutase 1 (SOD1) is secreted upon nutrient starvation, but the physiological relevance of this secretion is unclear. We now report that secreted SOD1 is functionally active. In addition, a secretome analysis has revealed number of other secreted cytoplasmic antioxidant enzymes, including both thioredoxins, (Trx1 and Trx2), and the peroxiredoxin Ahp1. Our data reveal that starvation triggers increased mitochondrial activity, leading to increased production of reactive oxygen species (ROS). Inhibiting mitochondrial activity or ROS, inhibited secretion of the antioxidants. We suggest elevated ROS that evade cytoplasmic antioxidants, can permeate across the plasma membrane to the extracellular space. Cells secrete antioxidants to prevent ROS-mediated damage to the extracellular space. These findings thus provide an understanding of why cells secrete signal sequence lacking proteins like SOD1 and the larger family of antioxidants.
Project description:Alzheimer’s disease (AD) is an unremitting neurodegenerative disorder characterized by cerebral amyloid-β (Aβ) accumulation and gradual decline in cognitive function. Changes in brain energy metabolism arise in the preclinical phase of AD, suggesting an important metabolic component of early AD pathology. Neurons and astrocytes function in close metabolic collaboration, which is essential for the recycling of neurotransmitters in the synapse. However, how this metabolic interplay may be affected during the early stages of AD development has not been sufficiently investigated. Here, we provide an integrative analysis of cellular metabolism during the early stages of Aβ accumulation in the cerebral cortex and hippocampus of the 5xFAD mouse model of AD. Our electrophysiological examination revealed an increase in spontaneous excitatory signaling in hippocampal brain slices of 5xFAD mice. This hyperactive neuronal phenotype coincided with decreased hippocampal TCA cycle metabolism mapped by stable 13C isotope tracing. Particularly, reduced astrocyte TCA cycle activity led to decreased glutamine synthesis, in turn hampering neuronal GABA synthesis in the 5xFAD hippocampus. In contrast, cerebral cortical slices of 5xFAD mice displayed an elevated capacity for oxidative glucose metabolism suggesting a metabolic compensation. When we explored the brain proteome and metabolome of the 5xFAD mice, we found limited changes, supporting that the functional metabolic disturbances between neurons and astrocytes are early events in AD pathology. In addition, we show that synaptic mitochondrial and glycolytic function was impaired selectively in the 5xFAD hippocampus, whereas non-synaptic mitochondrial function was maintained. These findings were supported by ultrastructural analyses demonstrating disruptions in mitochondrial morphology, particularly in the 5xFAD hippocampus. Collectively, our study reveals complex region and cell specific metabolic adaptations, in the early stages of amyloid pathology, which may be fundamental for the progressing synaptic dysfunction in AD.