Restructuring a Complex Genetic Function on Episomal Vectors in .

Genetic functions have evolved over long timescales and can be encoded by multiple genes dispersed in different locations in genomes, and although contemporary molecular biology enables control over single genes, more complex genetic functions remain challenging. Here, we study the restructuring and mobilization of a complex genetic function encoded by 10 genes, originally expressed from four operons and two loci on the genome. We observe subtle phenotypic differences and reduced fitness when expressed from episomal DNA and demonstrate that mutations in the transcriptional machinery are necessary for successful implementation in different bacteria.

Streamlined analysis of drug targets by proteome integral solubility alteration indicates organ-specific engagement.

Proteins are the primary targets of almost all small molecule drugs. However, even the most selectively designed drugs can potentially target several unknown proteins. Identification of potential drug targets can facilitate design of new drugs and repurposing of existing ones.

A seven-transmembrane methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in industrial biotechnology. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identify the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663.

Divergent Molecular and Cellular Responses to Low and High-Dose Ionizing Radiation.

Cancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low (LD; 0.1 Gy) or a high (HD; 1 Gy) dose of X-rays, we show that, although the DNA damage response (DDR) displayed dose proportionality, many other molecular and cellular responses did not.

A deeper look at carrier proteome effects for single-cell proteomics.

Multiplexing approaches using tandem mass tags with a carrier proteome to boost sensitivity have advanced single cell proteomics by mass spectrometry (SCoPE-MS). Here, we probe the carrier proteome effects in single cell proteomics with mixed species TMTpro-labeled samples. We demonstrate that carrier proteomes, while increasing overall identifications, dictate which proteins are identified.

Proteome and Phosphoproteome Changes Associated with Prognosis in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematological cancer that mainly affects the elderly. Although complete remission (CR) is achieved for the majority of the patients after induction and consolidation therapies, nearly two-thirds relapse within a short interval. Understanding biological factors that determine relapse has become of major clinical interest in AML.

The Role of TTP Phosphorylation in the Regulation of Inflammatory Cytokine Production by MK2/3.

Tristetraprolin (TTP) is an RNA-binding protein and an essential factor of posttranscriptional repression of cytokine biosynthesis in macrophages. Its activity is temporally inhibited by LPS-induced p38/MAPKAPK2/3-mediated phosphorylation, leading to a rapid increase in cytokine expression. We compared TTP expression and cytokine production in mouse bone marrow-derived macrophages of different genotypes: wild type, MAPKAP kinase 2 (MK2) deletion (MK2 knockout [KO]), MK2/3 double deletion (MK2/3 double KO [DKO]), TTP-S52A-S178A (TTPaa) knock-in, as well as combined MK2 KO/TTPaa and MK2/3 DKO/TTPaa.

Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9).

GIGYF1/2-Driven Cooperation between ZNF598 and TTP in Posttranscriptional Regulation of Inflammatory Signaling.

Inflammatory signaling is restricted through degradation and the translational repression of cytokine mRNAs. A key factor in this regulation is tristetraprolin (TTP), an RNA-binding protein (RBP) that recruits RNA-destabilizing factors and the translation inhibitory complex 4EHP-GIGYF1/2 to AU-rich element (ARE)-containing mRNAs. Here, we show that the RBP ZNF598 contributes to the same regulatory module in a TTP-like manner.

Protein Aggregation Capture on Microparticles Enables Multipurpose Proteomics Sample Preparation.

Universal proteomics sample preparation is challenging because of the high heterogeneity of biological samples. Here we describe a novel mechanism that exploits the inherent instability of denatured proteins for nonspecific immobilization on microparticles by protein aggregation capture. To demonstrate the general applicability of this mechanism, we analyzed phosphoproteomes, tissue proteomes, and interaction proteomes as well as dilute secretomes.

Proteogenomic Characterization of Patient-Derived Xenografts Highlights the Role of REST in Neuroendocrine Differentiation of Castration-Resistant Prostate Cancer.

Purpose: An increasing number of castration-resistant prostate cancer (CRPC) tumors exhibit neuroendocrine (NE) features. NE prostate cancer (NEPC) has poor prognosis, and its development is poorly understood. We applied mass spectrometry-based proteomics to a unique set of 17 prostate cancer patient-derived xenografts (PDX) to characterize the effects of castration , and the proteome differences between NEPC and prostate adenocarcinomas.

Limits for Resolving Isobaric Tandem Mass Tag Reporter Ions Using Phase-Constrained Spectrum Deconvolution.

A popular method for peptide quantification relies on isobaric labeling such as tandem mass tags (TMT), which enables multiplexed proteome analyses. Quantification is achieved by reporter ions generated by fragmentation in a tandem mass spectrometer. However, with higher degrees of multiplexing, the smaller mass differences between the reporter ions increase the mass resolving power requirements.

Large-Scale Phosphoproteomics Reveals Shp-2 Phosphatase-Dependent Regulators of Pdgf Receptor Signaling.

Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2, or Igf-1 and identified more than 40,000 phosphorylation sites, including many phosphotyrosine sites without additional enrichment. The analysis revealed RTK-specific regulation of hundreds of pTyr sites on key signaling molecules.

Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress.

The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers) has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress.

An Optimized Shotgun Strategy for the Rapid Generation of Comprehensive Human Proteomes.

This study investigates the challenge of comprehensively cataloging the complete human proteome from a single-cell type using mass spectrometry (MS)-based shotgun proteomics. We modify a classical two-dimensional high-resolution reversed-phase peptide fractionation scheme and optimize a protocol that provides sufficient peak capacity to saturate the sequencing speed of modern MS instruments. This strategy enables the deepest proteome of a human single-cell type to date, with the HeLa proteome sequenced to a depth of ∼584,000 unique peptide sequences and ∼14,200 protein isoforms (∼12,200 protein-coding genes).

The ubiquitin ligase Cullin5 regulates NDR1/STK38 stability and NF-κB transactivation.

SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates.

Offline High pH Reversed-Phase Peptide Fractionation for Deep Phosphoproteome Coverage.

Protein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental and metabolic stimuli, and are thus highly regulated. Therefore, studying the mechanism of regulation by phosphorylation, and pinpointing the exact site of phosphorylation on proteins is of high importance.

Principal component analysis of proteomics (PCAP) as a tool to direct metabolic engineering.

Targeted proteomics is a convenient method determining enzyme expression levels, but a quantitative analysis of these proteomic data has not been fully explored yet. Here, we present and demonstrate a computational tool (principal component analysis of proteomics, PCAP) that uses quantitative targeted proteomics data to guide metabolic engineering and achieve higher production of target molecules from heterologous pathways. The method is based on the application of principal component analysis to a collection of proteomics and target molecule production data to pinpoint specific enzymes that need to have their expression level adjusted to maximize production.

Rapid and deep proteomes by faster sequencing on a benchtop quadrupole ultra-high-field Orbitrap mass spectrometer.

Shotgun proteomics is a powerful technology for global analysis of proteins and their post-translational modifications. Here, we investigate the faster sequencing speed of the latest Q Exactive HF mass spectrometer, which features an ultra-high-field Orbitrap mass analyzer. Proteome coverage is evaluated by four different acquisition methods and benchmarked across three generations of Q Exactive instruments (ProteomeXchange data set PXD001305).

Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics.

Protein phosphorylation is an important post-translational modification (PTM) involved in embryonic development, adult homeostasis, and disease. Over the past decade, several advances have been made in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based technologies to identify thousands of phosphorylation sites. However, in-depth phosphoproteomics often require off-line enrichment and fractionation techniques.

A targeted proteomics toolkit for high-throughput absolute quantification of Escherichia coli proteins.

Transformation of engineered Escherichia coli into a robust microbial factory is contingent on precise control of metabolism. Yet, the throughput of omics technologies used to characterize cell components has lagged far behind our ability to engineer novel strains. To expand the utility of quantitative proteomics for metabolic engineering, we validated and optimized targeted proteomics methods for over 400 proteins from more than 20 major pathways in E.

Development of a native Escherichia coli induction system for ionic liquid tolerance.

The ability to solubilize lignocellulose makes certain ionic liquids (ILs) very effective reagents for pretreating biomass prior to its saccharification for biofuel fermentation. However, residual IL in the aqueous sugar solution can inhibit the growth and function of biofuel-producing microorganisms. In E.

Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly.

The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene).