Methylmalonic acidemia (MMA) is a rare inborn error of metabolism caused by deficiency of the methylmalonyl-CoA mutase (MUT) enzyme. Downstream MUT deficiency, methylmalonic acid accumulates together ...with toxic metabolites from propionyl-CoA and other compounds upstream of the block in the enzyme pathway. The presentation is with life-threatening acidosis, respiratory distress, brain disturbance, hyperammonemia, and ketosis. Survivors develop poorly understood multi-organ damage, notably to the brain and kidneys. The HEK 293 cell line was engineered by CRISPR/Cas9 technology to knock out the
gene (MUT-KO). Shotgun label-free quantitative proteomics and bioinformatics analyses revealed potential damaging biological processes in MUT-deficient cells. MUT-KO induced alteration of cellular architecture and morphology, and ROS overproduction. We found the alteration of proteins involved in cytoskeleton and cell adhesion organization, cell trafficking, mitochondrial, and oxidative processes, as validated by the regulation of VIM, EXT2, SDC2, FN1, GLUL, and CHD1. Additionally, a cell model of MUT-rescuing was developed in order to control the specificity of MUT-KO effects. Globally, the proteomic landscape of MUT-KO suggests the cell model to have an increased susceptibility to propionate- and H
O
-induced stress through an impairment of the mitochondrial functionality and unbalances in the oxidation-reduction processes.
The enzyme fructose-bisphosphate aldolase occupies a central position in glycolysis and gluconeogenesis pathways. Beyond its housekeeping role in metabolism, fructose-bisphosphate aldolase has been ...involved in additional functions and is considered as a potential target for drug development against pathogenic bacteria. Here, we address the role of fructose-bisphosphate aldolase in the bacterial pathogen Francisella novicida. We demonstrate that fructose-bisphosphate aldolase is important for bacterial multiplication in macrophages in the presence of gluconeogenic substrates. In addition, we unravel a direct role of this metabolic enzyme in transcription regulation of genes katG and rpoA, encoding catalase and an RNA polymerase subunit, respectively. We propose a model in which fructose-bisphosphate aldolase participates in the control of host redox homeostasis and the inflammatory immune response.The enzyme fructose-bisphosphate aldolase (FBA) plays central roles in glycolysis and gluconeogenesis. Here, Ziveri et al. show that FBA of the pathogen Francisella novicida acts, in addition, as a transcriptional regulator and is important for bacterial multiplication in macrophages.
Bacterial pathogens adapt and replicate within host cells, while host cells develop mechanisms to eliminate them. Using a dual proteomic approach, we characterized the intra-macrophage proteome of ...the facultative intracellular pathogen, Francisella novicida. More than 900 Francisella proteins were identified in infected macrophages after a 10-h infection. Biotin biosynthesis-related proteins were upregulated, emphasizing the role of biotin-associated genes in Francisella replication. Conversely, proteins encoded by the Francisella pathogenicity island (FPI) were downregulated, supporting the importance of the F. tularensis Type VI Secretion System for vacuole escape, not cytosolic replication. In the host cell, over 300 proteins showed differential expression among the 6200 identified during infection. The most upregulated host protein was cis-aconitate decarboxylase IRG1, known for itaconate production with antimicrobial properties in Francisella. Surprisingly, disrupting IRG1 expression did not impact Francisella's intracellular life cycle, suggesting redundancy with other immune proteins or inclusion in larger complexes. Over-representation analysis highlighted cell-cell contact and actin polymerization in macrophage deregulated proteins. Using flow cytometry and live cell imaging, we demonstrated that merocytophagy involves diverse cell-to-cell contacts and actin polymerization-dependent processes. These findings lay the groundwork for further exploration of merocytophagy and its molecular mechanisms in future research.Data are available via ProteomeXchange with identifier PXD035145.
Biolayer interferometry (BLI) is a technology which allows to study the affinity between two interacting macro‐molecules and to visualize their kinetic of interaction in real time. In this work, we ...combine BLI interaction measurement with mass spectrometry in order to identify the proteins interacting with the bait. We provide for the first time the proof of concept of the feasibility of BLI‐MS in complex biological mixtures.
STK38 (also known as NDR1) is a Hippo pathway serine/threonine protein kinase with multifarious functions in normal and cancer cells. Using a context‐dependent proximity‐labeling assay, we identify ...more than 250 partners of STK38 and find that STK38 modulates its partnership depending on the cellular context by increasing its association with cytoplasmic proteins upon nutrient starvation‐induced autophagy and with nuclear ones during ECM detachment. We show that STK38 shuttles between the nucleus and the cytoplasm and that its nuclear exit depends on both XPO1 (aka exportin‐1, CRM1) and STK38 kinase activity. We further uncover that STK38 modulates XPO1 export activity by phosphorylating XPO1 on serine 1055, thus regulating its own nuclear exit. We expand our model to other cellular contexts by discovering that XPO1 phosphorylation by STK38 regulates also the nuclear exit of Beclin1 and YAP1, key regulator of autophagy and transcriptional effector, respectively. Collectively, our results reveal STK38 as an activator of XPO1, behaving as a gatekeeper of nuclear export. These observations establish a novel mechanism of XPO1‐dependent cargo export regulation by phosphorylation of XPO1's C‐terminal auto‐inhibitory domain.
Synopsis
Cytoplasmic accumulation of STK38 kinase is essential for starvation‐induced autophagy. STK38 phosphorylates and activates the nuclear export factor XPO1, thereby supporting the shuttling of the autophagy regulator Beclin1 and other XPO1 cargoes to the cytoplasm.
XPO1 is a substrate for the STK38 kinase.
XPO1 is activated by this phosphorylation event on Ser1055.
STK38 behaves as a regulator of XPO1 activity, which is inactive in absence of phosphorylation of its Ser1055.
Cytoplasmic accumulation of STK38 kinase is essential for starvation‐induced autophagy. STK38 phosphorylates and activates the nuclear export factor XPO1, thereby supporting the shuttling of the autophagy regulator Beclin1 and other XPO1 cargoes to the cytoplasm.
Metabolic pathways are now considered as intrinsic virulence attributes of pathogenic bacteria and thus represent potential targets for antibacterial strategies. Here we focused on the role of the ...pentose phosphate pathway (PPP) and its connections with other metabolic pathways in the pathophysiology of
Francisella novicida
. The involvement of the PPP in the intracellular life cycle of
Francisella
was first demonstrated by studying PPP inactivating mutants. Indeed, we observed that inactivation of the
tktA
,
rpiA
or
rpe
genes severely impaired intramacrophage multiplication during the first 24 hours. However, time-lapse video microscopy demonstrated that
rpiA
and
rpe
mutants were able to resume late intracellular multiplication. To better understand the links between PPP and other metabolic networks in the bacterium, we also performed an extensive proteo-metabolomic analysis of these mutants. We show that the PPP constitutes a major bacterial metabolic hub with multiple connections to glycolysis, the tricarboxylic acid cycle and other pathways, such as fatty acid degradation and sulfur metabolism. Altogether our study highlights how PPP plays a key role in the pathogenesis and growth of
Francisella
in its intracellular niche.
Plasma proteomics holds immense potential for clinical research and biomarker discovery, serving as a non-invasive "liquid biopsy" for tissue sampling. Mass spectrometry (MS)-based proteomics, thanks ...to improvement in speed and robustness, emerges as an ideal technology for exploring the plasma proteome for its unbiased and highly specific protein identification and quantification. Despite its potential, plasma proteomics is still a challenge due to the vast dynamic range of protein abundance, hindering the detection of less abundant proteins. Different approaches can help overcome this challenge. Conventional depletion methods face limitations in cost, throughput, accuracy, and off-target depletion. Nanoparticle-based enrichment shows promise in compressing dynamic range, but cost remains a constraint. Enrichment strategies for extracellular vesicles (EVs) can enhance plasma proteome coverage dramatically, but current methods are still too laborious for large series. Neat plasma remains popular for its cost-effectiveness, time efficiency, and low volume requirement. We used a test set of 33 plasma samples for all evaluations. Samples were digested using S-Trap and analyzed on Evosep One and nanoElute coupled to a timsTOF Pro using different elution gradients and ion mobility ranges. Data were mainly analyzed using library-free searches using DIA-NN. This study explores ways to improve proteome coverage in neat plasma both in MS data acquisition and MS data analysis. We demonstrate the value of sampling smaller hydrophilic peptides, increasing chromatographic separation, and using library-free searches. Additionally, we introduce the EV boost approach, that leverages on the extracellular vesicle fraction to enhance protein identification in neat plasma samples. Globally, our optimized analysis workflow allows the quantification of over 1000 proteins in neat plasma with a 24SPD throughput. We believe that these considerations can be of help independently of the LC-MS platform used.
The analysis of T cell lipid raft proteome is challenging due to the highly dynamic nature of rafts and the hydrophobic character of raft-resident proteins. We explored an innovative strategy for ...bottom-up lipid raftomics based on suspension-trapping (S-Trap) sample preparation. Mouse T cells were prepared from splenocytes by negative immunoselection, and rafts were isolated by a detergent-free method and OptiPrep gradient ultracentrifugation. Microdomains enriched in flotillin-1, LAT, and cholesterol were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. Using this method, we identified 2,680 proteins in the raft-rich fraction and established a database of 894 T cell raft proteins. We then performed a differential analysis on the raft-rich fraction from nonstimulated versus anti-CD3/CD28 T cell receptor (TCR)-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other. For the first time, we performed a proteomic analysis on rafts from ex vivo T cells obtained from individual mice, before and after TCR activation. This work demonstrates that the proposed method utilizing an S-Trap-based approach for sample preparation increases the specificity and sensitivity of lipid raftomics.
Cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) are pulmonary genetic disorders associated with inflammation and heterogeneous progression of the lung disease. We hypothesized that ...respiratory exosomes, nanovesicles circulating in the respiratory tract, may be involved in the progression of inflammation-related lung damage. We compared proteomic content of respiratory exosomes isolated from bronchoalveolar lavage fluid in CF and PCD to asthma (A), a condition also associated with inflammation but with less severe lung damage.
BALF were obtained from 3 CF, 3 PCD and 6 A patients. Exosomes were isolated from BALF by ultracentrifugations and characterized using immunoelectron microscopy and western-blot. Exosomal protein analysis was performed by high-resolution mass spectrometry using label-free quantification.
Exosome enrichment was validated by electron microscopy and immunodetection of CD9, CD63 and ALIX. Mass spectrometry analysis allowed the quantification of 665 proteins, of which 14 were statistically differential according to the disease. PCD and CF exosomes contained higher levels of antioxidant proteins (Superoxide-dismutase, Glutathione peroxidase-3, Peroxiredoxin-5) and proteins involved in leukocyte chemotaxis. All these proteins are known activators of the NF-KappaB pathway.
Our results suggest that respiratory exosomes are involved in the pro-inflammatory propagation during the extension of CF or PCD lung diseases.
The mechanism of local propagation of lung disease in cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) is not clearly understood. Differential Proteomic profiles of exosomes isolated from BAL from CF, PCD and asthmatic patients suggest that they carry pro-inflammatory proteins that may be involved in the progression of lung damage.
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•Exosomes were detected in BALF from CF, PCD and asthmatic patients•The exosomes from PCD and CF patients differed significantly from those isolated from asthmatic patients.•PCF and CF exosomes contained higher levels of antioxidant proteins
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