Intricate Crosstalk Between Lipopolysaccharide, Phospholipid and Fatty Acid Metabolism in Escherichia coli Modulates Proteolysis of LpxC

Lipopolysaccharides (LPS) in the outer membrane of Gram-negative bacteria provide the first line of defense against antibiotics and other harmful compounds. LPS biosynthesis critically depends on LpxC catalyzing the first committed enzyme in this process. In Escherichia coli, the cellular concentration of LpxC is adjusted in a growth rate-dependent manner by the FtsH protease making sure that LPS biosynthesis is coordinated with the cellular demand. As a result, LpxC is stable in fast-growing cells and prone to degradation in slow-growing cells. One of the factors involved in this process is the alarmone guanosine tetraphosphate (ppGpp) but previous studies suggested the involvement of yet unknown factors in LpxC degradation. We established a quantitative proteomics approach aiming at the identification of proteins that are associated with LpxC and/or FtsH at high or low growth rates. The identification of known LpxC and FtsH interactors validated our approach. A number of proteins involved in fatty acid biosynthesis and degradation, including the central regulator FadR, were found in the LpxC and/or FtsH interactomes. Another protein associated with LpxC and FtsH was WaaH, a LPS-modifying enzyme. When overproduced, several members of the LpxC/FtsH interactomes were able to modulate LpxC proteolysis. Our results go beyond the previously established link between LPS and phospholipid biosynthesis and uncover a far-reaching network that controls LPS production by involving multiple enzymes in fatty acid metabolism, phospholipid biosynthesis and LPS modification

LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear

Regulation and function of the phytochrome system in Pseudomonas aeruginosa\textit {Pseudomonas aeruginosa}

Phytochromes are red/far-red light photoreceptors found in plants and have recently also been discovered in bacteria. Biochemical analysis established that the genes $\it {bphO}$ and $\it {bphP}$ display the two necessary phytochrome components in the opportunistic pathogen $\textit {Pseudomonas aeruginosa: bphO}$ encoding a heme oxygenase that produces the chromophore biliverdin IX$\alpha$ and $\it {bphP}$ encoding the apo-phytochrome. Transcriptional analyses established that both genes form a bicistronic operon. Expression of the $\it {bphOP}$ operon was shown to be induced in the stationary phase dependent on the alternative sigma factor, RpoS. Interestingly, the loss of BphO but not BphP led to pronounced phenotypes in the stationary phase suggesting an additional role of BphO besides providing the chromophore to BphP. Transcriptome and proteom studies on the other hand revealed that BphP is involved in the regulation of various gene clusters and pointed to a link of BphP function with the $\textit {P. aeruginosa}$ quorum sensing system

Statically Adsorbed Coatings for High Separation Efficiency and Resolution in CE–MS Peptide Analysis: Strategies and Implementation

Coatings are necessary to prevent protein and peptide adsorption to the capillary surface and obtain high intermediate precision. In this protocol, we first present our basic strategy to address peptide separation using three different coatings: one neutral and two cationic coatings, the latter largely differing in their induced electroosmotic mobility. In detail, we will describe how we apply the statically adsorbed coatings to obtain very high plate numbers and high repeatability.With some model examples, we clearly describe the scope of the method for the analysis of peptide samples: tryptic digests are addressed as well as small glycoproteins and glycopeptides largely differing in their effective electrophoretic mobility. We also show that the method is suitable for a fast screening of peptide samples despite a high matrix load comprising of up to 500 mmol/L sodium chloride. We demonstrate that this basic CE-MS method is rather independent of the polarity of the analytes with a very fast near-baseline separation of very hydrophobic Aβ peptides related to the onset of Alzheimer's disease. These examples will give an impression, which coating is most suitable for a specific analytical application.Special attention is paid to difficult aspects of the coating procedure and the CE-MS method, e.g., the potential of cross-contamination when changing the coatings

Dataset containing physiological amounts of spike-in proteins into murine C2C12 background as a ground truth quantitative LC-MS/MS reference

In this article, we present a data dependent acquisition (DDA) dataset which was generated as a reference and ground truth quantitative dataset. While initially used to compare samples measured with DDA and data independent acquisition (DIA) (Barkovits et al., 2020), the presented dataset holds potential value as a benchmark reference for any workflows working on DDA data. The entire dataset consists of 15 LC-MS/MS measurements composed of five distinct spike-in-states, each with three replicates. To generate the data set, a C2C12 (immortalized mouse myoblast) cell lysate was used as a complex background for five different states which were simulated by spiking 13 defined proteins at different concentrations. For this purpose, the cell lysate was used in a constant amount of 20 µg for all samples and different amounts of the 13 selected proteins ranging from 0.1 to 10 pmol were added, reflecting physiological amounts of proteins. Afterwards, all samples were tryptically digested using the same method. From each sample 200 ng tryptic peptides were measured in triplicates on a Q Exactive HF (Thermo Fisher Scientific). The mass range for MS1 was set to 350–1400 m/z with a resolution of 60,000 at 200 m/z. HCD fragmentation of the Top10 abundant precursor ions was performed at 27% NCE. The fragment analysis (MS2) was performed with a resolution of 30,000 at 200 m/z

Metabolic profiling of body fluids and multivariate data analysis

Metabolome analyses of body fluids are challenging due pre-analytical variations, such as pre-processing delay and temperature, and constant dynamical changes of biochemical processes within the samples. Therefore, proper sample handling starting from the time of collection up to the analysis is crucial to obtain high quality samples and reproducible results. A metabolomics analysis is divided into 4 main steps: 1) Sample collection, 2) Metabolite extraction, 3) Data acquisition and 4) Data analysis. Here, we describe a protocol for gas chromatography coupled to mass spectrometry (GC–MS) based metabolic analysis for biological matrices, especially body fluids. This protocol can be applied on blood serum/plasma, saliva and cerebrospinal fluid (CSF) samples of humans and other vertebrates. It covers sample collection, sample pre-processing, metabolite extraction, GC–MS measurement and guidelines for the subsequent data analysis. Advantages of this protocol include: • Robust and reproducible metabolomics results, taking into account pre-analytical variations that may occur during the sampling process • Small sample volume required • Rapid and cost-effective processing of biological samples • Logistic regression based determination of biomarker signatures for in-depth data analysi

Advanced Fiber Type-Specific Protein Profiles Derived from Adult Murine Skeletal Muscle

Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set

The proteome of neuromelanin granules in dementia with Lewy bodies

Neuromelanin granules (NMGs) are organelle-like structures present in the human substantia nigra pars compacta. In addition to neuromelanin, NMGs contain proteins, lipids and metals. As NMG-containing dopaminergic neurons are preferentially lost in Parkinson’s disease and dementia with Lewy bodies (DLB), it is assumed that NMGs may play a role in neurodegenerative processes. Until now, this role is not completely understood and needs further investigation. We therefore set up an exploratory proteomic study to identify differences in the proteomic profile of NMGs from DLB patients (n = 5) compared to healthy controls (CTRL, n = 5). We applied a laser microdissection and mass-spectrometry-based approach, in which we used targeted mass spectrometric experiments for validation. In NMG-surrounding (SN$_{Surr.}$) tissue of DLB patients, we found evidence for ongoing oxidative damage and an impairment of protein degradation. As a potentially disease-related mechanism, we found α-synuclein and protein S100A9 to be enriched in NMGs of DLB cases, while the abundance of several ribosomal proteins was significantly decreased. As S100A9 is known to be able to enhance the formation of toxic α-synuclein fibrils, this finding points towards an involvement of NMGs in pathogenesis, however the exact role of NMGs as either neuroprotective or neurotoxic needs to be further investigated. Nevertheless, our study provides evidence for an impairment of protein degradation, ongoing oxidative damage and accumulation of potentially neurotoxic protein aggregates to be central mechanisms of neurodegeneration in DLB

Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM) on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4) comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m.)) deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s) for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures