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Cell Oct 2015The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular...
The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.
Topics: Cell Line; Chromosomes, Artificial, Bacterial; Humans; Protein Interaction Mapping; Proteomics
PubMed: 26496610
DOI: 10.1016/j.cell.2015.09.053 -
MBio Aug 2020The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the...
The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a few studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically based real-time microtiter plate readout with a matrix-like heat map of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of extraintestinal pathogenic (ExPEC) with antibiotic levels blanketing the MIC across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, two different related phages that differed in their burst sizes produced drastically different synograms. Collectively, these data suggest lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria while also synergizing with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments. Bacteriophage (phage) therapy is a promising approach to combat the rise of multidrug-resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment.
Topics: Anti-Bacterial Agents; Bacteriophages; Drug Antagonism; Drug Resistance, Multiple, Bacterial; Drug Synergism; Escherichia coli; Humans; Microbial Sensitivity Tests; Phage Therapy
PubMed: 32753497
DOI: 10.1128/mBio.01462-20 -
Cell Reports Sep 2014Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics,...
Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes.
Topics: Algorithms; HeLa Cells; Humans; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Proteome; Sequence Analysis, Protein; Serine; Signal Transduction; Threonine; Tyrosine
PubMed: 25159151
DOI: 10.1016/j.celrep.2014.07.036 -
Trends in Biochemical Sciences Feb 2019The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis has been speculated for over 60 years.... (Review)
Review
The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis has been speculated for over 60 years. Recent advances in mass spectrometry (MS) and high-throughput analysis have begun to identify and characterize distinct ribosome stoichiometry in yeast and mammalian systems. In addition to RP stoichiometry, ribosomes host a vast array of protein modifications, effectively expanding the number of human RPs from 80 to many thousands of distinct proteoforms. Is it possible that these proteoforms combine to function as a 'ribosome code' to tune protein synthesis? We outline the specific benefits that translational regulation by specialized ribosomes can offer and discuss the means and methodologies available to correlate and characterize RP stoichiometry with function. We highlight previous research with a focus on formulating hypotheses that can guide future experiments and crack the ribosome code.
Topics: Animals; High-Throughput Screening Assays; Humans; Mass Spectrometry; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae
PubMed: 30473427
DOI: 10.1016/j.tibs.2018.10.009 -
Chemical Reviews Apr 2022Native mass spectrometry (MS) is aimed at preserving and determining the native structure, composition, and stoichiometry of biomolecules and their complexes from... (Review)
Review
Native mass spectrometry (MS) is aimed at preserving and determining the native structure, composition, and stoichiometry of biomolecules and their complexes from solution after they are transferred into the gas phase. Major improvements in native MS instrumentation and experimental methods over the past few decades have led to a concomitant increase in the complexity and heterogeneity of samples that can be analyzed, including protein-ligand complexes, protein complexes with multiple coexisting stoichiometries, and membrane protein-lipid assemblies. Heterogeneous features of these biomolecular samples can be important for understanding structure and function. However, sample heterogeneity can make assignment of ion mass, charge, composition, and structure very challenging due to the overlap of tens or even hundreds of peaks in the mass spectrum. In this review, we cover data analysis, experimental, and instrumental advances and strategies aimed at solving this problem, with an in-depth discussion of theoretical and practical aspects of the use of available deconvolution algorithms and tools. We also reflect upon current challenges and provide a view of the future of this exciting field.
Topics: Algorithms; Ligands; Mass Spectrometry; Membrane Lipids; Proteins
PubMed: 34470219
DOI: 10.1021/acs.chemrev.1c00696 -
Current Genetics Dec 2021Cellular systems depend on multiprotein complexes whose functionalities require defined stoichiometries of subunit proteins. Proper stoichiometry is achieved by... (Review)
Review
Cellular systems depend on multiprotein complexes whose functionalities require defined stoichiometries of subunit proteins. Proper stoichiometry is achieved by controlling the amount of protein synthesis and degradation even in the presence of genetic perturbations caused by changes in gene dosage. As a consequence of increased gene copy number, excess subunits unassembled into the complex are synthesized and rapidly degraded by the ubiquitin-proteasome system. This mechanism, called protein-level dosage compensation, is widely observed not only under such perturbed conditions but also in unperturbed physiological cells. Recent studies have shown that recognition of unassembled subunits and their selective degradation are intricately regulated. This review summarizes the nature, strategies, and increasing complexity of protein-level dosage compensation and discusses possible mechanisms for controlling proteome stoichiometry in multiple layers of biological processes.
Topics: Fungal Proteins; Gene Expression Regulation, Fungal; Models, Biological; Multiprotein Complexes; Protein Binding; Protein Biosynthesis; Proteolysis; Proteome; Yeasts
PubMed: 34382105
DOI: 10.1007/s00294-021-01205-z -
Journal of Neurochemistry May 2021Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family... (Review)
Review
Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.
Topics: Animals; Humans; Neurotransmitter Transport Proteins
PubMed: 32767560
DOI: 10.1111/jnc.15145 -
Proceedings of the National Academy of... Aug 2022Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and...
Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits. By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide-binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.
Topics: Humans; Mass Spectrometry; Microscopy, Electron; Protein Domains; Shelterin Complex
PubMed: 35881804
DOI: 10.1073/pnas.2201662119 -
Molecules (Basel, Switzerland) Apr 2021A modification of Au(pMBA) that incorporates one diglyme ligand as a direct synthetic product is reported. Notably the expected statistical production of clusters...
A modification of Au(pMBA) that incorporates one diglyme ligand as a direct synthetic product is reported. Notably the expected statistical production of clusters containing other ligand stoichiometries is not observed. This Au(pMBA)diglyme product is characterized by electrospray ionization mass spectrometry (ESI-MS) and optical spectroscopy. Thiolate for thiolate ligand exchange proceeds on this cluster, whereas thiolate for diglyme ligand exchange does not.
PubMed: 33924805
DOI: 10.3390/molecules26092562 -
Materials (Basel, Switzerland) Sep 2023P-type Cu-Se thin films were deposited on glass substrates at room temperature using radio frequency magnetron sputtering by a single multi-component CuSe target. When...
P-type Cu-Se thin films were deposited on glass substrates at room temperature using radio frequency magnetron sputtering by a single multi-component CuSe target. When using a multi-component target, the impact of the sputtering power on the homogeneity and stoichiometry within the thin films should be investigated in the depth direction to demonstrate a secondary effect on the electrical and optical properties of the thin films. Systematic characterization of the Cu-Se thin films, including the morphology, microstructure, chemical composition, and depth-directional chemical bonding state and defect structure of the thin films, revealed that the sputtering power played an important role in the homogeneity and stoichiometry of the thin films. At very low and very high sputtering power levels, the Cu-Se thin films exhibited more deviations from stoichiometry, while an optimized sputtering power resulted in more homogenous thin films with improved stoichiometry across the entire thin film thickness in the X-ray photoelectron spectroscopy depth profile, despite showing Se deficiency at all depths. A rapid decrease in carrier concentration, indicating a reduction in the net effect of total defects, was obtained at the optimized sputtering power with less deviation from stoichiometry in the Cu-Se thin films and the closest stoichiometric ratio at an intermediate depth.
PubMed: 37763365
DOI: 10.3390/ma16186087