Targeted proteomics, which includes parallel reaction monitoring (PRM), is typically utilized for more precise detection and quantitation of key proteins and/or pathways derived from complex discovery proteomics datasets. Initial discovery-based analysis using data independent acquisition (DIA) can obtain deep proteome coverage with low data missingness while targeted PRM assays can provide additional benefits in further eliminating missing data and optimizing measurement precision. However, PRM method development from bioinformatic predictions can be tedious and time-consuming because of the DIA output complexity.
View Article and Find Full Text PDFAccurate and reliable detection of fungal pathogens presents an important hurdle to manage infections, especially considering that fungal pathogens, including the globally important human pathogen, , have adapted diverse mechanisms to survive the hostile host environment and moderate virulence determinant production during coinfections. These pathogen adaptations present an opportunity for improvements (e.g.
View Article and Find Full Text PDFis an important pathogen often associated with hospital-acquired infections and chronic lung infections in people with cystic fibrosis. possesses a wide array of intrinsic and adaptive mechanisms of antibiotic resistance, and the regulation of these mechanisms is complex. Label-free quantitative proteomics is a powerful tool to compare susceptible and resistant strains of bacteria and their responses to antibiotic treatments.
View Article and Find Full Text PDFHere, we present a protocol using MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) platform to investigate changes of the protein synthesis machinery in U87MG glioblastoma cells in response to the rocaglate silvestrol. This protocol describes steps to perform SILAC (stable isotope labeling by amino acids in cell culture), ribosome density fractionation, protein isolation, and mass spectrometry analysis. This approach can be applied to study any adaptive remodeling of protein synthesis machineries.
View Article and Find Full Text PDFThe interactions between a host and microbe drive the health and disease status of the host. Of importance is the cause of dysbiosis in the presence of a pathogen, and critically, the relationship between the host and pathogen may evolve over time through response and adaptation. For immunocompromised individuals, dual infections are prevalent and contribute to disease severity and treatment options.
View Article and Find Full Text PDFTransient expression of recombinant proteins in plants is being used as a platform for production of therapeutic proteins. Benefits of this system include a reduced cost of drug development, rapid delivery of new products to the market, and an ability to provide safe and efficacious medicines for diseases. Although plant-based production systems offer excellent potential for therapeutic protein production, barriers, such as plant host defense response, exist which negatively impact the yield of product.
View Article and Find Full Text PDFPhosphorylation is a key post-translational modification central to the biological behavior of proteins. This reversible modification specifically regulates cell signaling mechanisms to control survival and growth. Moreover, microbial pathogens, including both fungi and bacteria, rely on this modification to coordinate protein production and functioning during infection and dissemination within a host.
View Article and Find Full Text PDFTactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin.
View Article and Find Full Text PDFPurpose: Comparative genomics and phenotypic assays have shown that antibiotic resistance profiles differ among clinical isolates of Pseudomonas aeruginosa and that genotype-phenotype associations are difficult to establish for resistance phenotypes based on these comparisons alone.
Experimental Design: Here, we used label-free quantitative proteomics to compare two isolates of the Liverpool Epidemic Strain (LES) of P. aeruginosa, LESlike1 and LESB58, and the common laboratory strain P.
Inaccurate expression of the genetic code, also known as mistranslation, is an emerging paradigm in microbial studies. Growing evidence suggests that many microbial pathogens can deliberately mistranslate their genetic code to help invade a host or evade host immune responses. However, discovering different capacities for deliberate mistranslation remains a challenge because each group of pathogens typically employs a unique mistranslation mechanism.
View Article and Find Full Text PDFTranslatome reprogramming is a primary determinant of protein levels during stimuli adaptation. This raises the question: what are the translatome remodelers that reprogram protein output to activate biochemical adaptations. Here, we identify a translational pathway that represses metabolism to safeguard genome integrity.
View Article and Find Full Text PDFSerine hydroxymethyltransferase 2 (SHMT2) converts serine plus tetrahydrofolate (THF) into glycine plus methylene-THF and is upregulated at the protein level in lung and other cancers. In order to better understand the role of SHMT2 in cancer a model system of HeLa cells engineered for inducible over-expression or knock-down of SHMT2 was characterized for cell proliferation and changes in metabolites and proteome as a function of SHMT2. Ectopic over-expression of SHMT2 increased cell proliferation in vitro and tumor growth in vivo.
View Article and Find Full Text PDFMass spectrometry-based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly Pseudomonas aeruginosa, and the formation of biofilms. The interplay between P.
View Article and Find Full Text PDFProtein expression evolves under greater evolutionary constraint than mRNA levels, and translation efficiency represents a primary determinant of protein levels during stimuli adaptation. This raises the question as to the translatome remodelers that titrate protein output from mRNA populations. Here, we uncover a network of RNA-binding proteins (RBPs) that enhances the translation efficiency of glycolytic proteins in cells responding to oxygen deprivation.
View Article and Find Full Text PDFThe surface of nanoparticles changes immediately after intravenous injection because blood proteins adsorb on the surface. How this interface changes during circulation and its impact on nanoparticle distribution within the body is not understood. Here, we developed a workflow to show that the evolution of proteins on nanoparticle surfaces predicts the biological fate of nanoparticles .
View Article and Find Full Text PDFThe balance between comprehensively analyzing the proteome and using valuable mass spectrometry time is a genuine challenge in the field of proteomics. Multidimensional fractionation strategies have significantly increased proteome coverage, but often at the cost of increased mass analysis time, despite advances in mass spectrometer acquisition rates. Recently, the Evosep One liquid chromatography system was shown to analyze peptide samples in a high-throughput manner without sacrificing in-depth proteomics coverage.
View Article and Find Full Text PDFNanoparticles are engineered from materials such as metals, polymers, and different carbon allotropes that do not exist within the body. Exposure to these exogenous compounds raises concerns surrounding toxicity, inflammation, and immune activation. These responses could potentially be mitigated by synthesizing nanoparticles directly from molecules derived from the host.
View Article and Find Full Text PDFIn most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice.
View Article and Find Full Text PDFIn Escherichia coli, formation of new cells is mediated by the elongasome and divisome that govern cell elongation and septation, respectively. Proper transition between these events is essential to ensure viable progeny are produced; however, the components of each complex responsible for transmission of the cell signal to shift from elongation to septation are unclear. Recently, a region within the N-terminal domain of the essential divisome protein FtsK (FtsK) was identified that points to a key role for FtsK as a checkpoint of cell envelope remodeling during division.
View Article and Find Full Text PDFThe eukaryotic translation initiation factor 5B (eIF5B) is a homolog of IF2, an ancient translation factor that enables initiator methionine-tRNAi (met-tRNAi) loading on prokaryotic ribosomes. While it can be traced back to the last universal common ancestor, eIF5B is curiously dispensable in modern aerobic yeast and mammalian cells. Here, we show that eIF5B is an essential element of the cellular hypoxic cap-dependent protein synthesis machinery.
View Article and Find Full Text PDFThe capacity to coordinate environmental sensing with initiation of cellular responses underpins microbial survival and is crucial for virulence and stress responses in microbial pathogens. Here we define circuitry that enables the fungal pathogen Candida albicans to couple cell cycle dynamics with responses to cell wall stress induced by echinocandins, a front-line class of antifungal drugs. We discover that the C.
View Article and Find Full Text PDFAxon degeneration is an early event and pathological in neurodegenerative conditions and nerve injuries. To discover agents that suppress neuronal death and axonal degeneration, we performed drug screens on primary rodent neurons and identified the pan-kinase inhibitor foretinib, which potently rescued sympathetic, sensory, and motor and SOD1 mutant neurons from trophic factor withdrawal-induced degeneration. By using primary sympathetic neurons grown in mass cultures and Campenot chambers, we show that foretinib protected neurons by suppressing both known degenerative pathways and a new pathway involving unliganded TrkA and transcriptional regulation of the proapoptotic BH3 family members BimEL, Harakiri,and Puma, culminating in preservation of mitochondria in the degenerative setting.
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