Dual proteomics of infected macrophages reveal bacterial and host players involved in the Francisella intracellular life cycle and cell to cell dissemination by merocytophagy.

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.

Role of KCNK3 Dysfunction in Dasatinib-associated PAH and Endothelial Cell Dysfunction.

Pulmonary arterial hypertension (PAH) is severe cardiopulmonary disease that may be triggered by exposure to drugs such as dasatinib or facilitated by genetic predispositions. The incidence of dasatinib-associated PAH is estimated at 0.45%, suggesting individual predispositions. The mechanisms of dasatinib-associated PAH are still incomplete. We discovered a KCNK3 gene (coding for outward K+ channel) variant in a patient with dasatinib-associated PAH, and we investigated the impact of this variant on KCNK3 function. Additionally, we assessed the effects of dasatinib exposure on KCNK3 expression. In control-human in pulmonary arterial smooth muscle cells (hPASMCs) and pulmonary endothelial cells (hPECs), we evaluated the consequence of KCNK3 knockdown on cell migration, mitochondrial membrane potential, ATP production, and in vitro tube formation. Using mass spectrometry, we determined the KCNK3 interactome. Patch-clamp revealed that the KCNK3 variant represents a loss-of-function variant. Dasatinib contributed to pulmonary artery constriction by decreasing KCNK3 function and expression. In control-hPASMCs, KCNK3 knockdown promotes mitochondrial membrane depolarization and glycolytic shift. Dasatinib exposure or KCNK3 knockdown reduced the number of caveolae in hPECs. Moreover, KCNK3 knockdown in control-hPECs reduced migration, proliferation, and in vitro tubulogenesis. Using proximity labeling and mass spectrometry, we identified the KCNK3 interactome, revealing that KCNK3 interacts with various proteins across different cellular compartments. We identified a novel pathogenic variant in the KCNK3 and showed that dasatinib downregulates KCNK3, emphasizing the relationship between dasatinib-associated PAH and KCNK3 dysfunction. We demonstrated that loss of KCNK3-dependent signaling contributes to endothelial dysfunction in PAH and glycolytic switch of hPASMCs.

Effect of urine alkalization on urinary inflammatory markers in cystinuric patients.

Background: Cystinuria is associated with a high prevalence of chronic kidney disease (CKD). We previously described a urinary inflammatory-protein signature (UIS), including 38 upregulated proteins, in cystinuric patients (Cys-patients), compared with healthy controls (HC). This UIS was higher in Cys-patients with CKD. In the present observational study, we aimed to investigate the UIS in Cys-patients without CKD and patients with calcium nephrolithiasis (Lith-patients), versus HC and the effect of urine alkalization on the UIS of Cys-patients. Methods: UIS was evaluated by nano-liquid chromatography coupled to high-resolution mass spectrometry in adult HC, Lith-patients and non-treated Cys-patients with an estimated glomerular filtration rate >60 mL/min/1.73 m2, and after a 3-month conventional alkalizing treatment in Cys-patients. Results: Twenty-one Cys-patients [12 men, median age (interquartile range) 30.0 (25.0-44.0) years], 12 Lith-patients [8 men, 46.2 (39.5-54.2) years] and 7 HC [2 men, 43.1 (31.0-53.9) years] were included. Among the 38 proteins upregulated in our previous work, 11 proteins were also upregulated in Cys-patients compared with HC in this study (5 circulating inflammatory proteins and 6 neutrophil-derived proteins). This UIS was also found in some Lith-patients. Using this UIS, we identified two subclusters of Cys-patients (5 with a very high/high UIS and 16 with a moderate/low UIS). In the Cys-patients with very high/high UIS, urine alkalization induced a significant decrease in urinary neutrophil-derived proteins. Conclusion: A high UIS is present in some Cys-patients without CKD and decreases under alkalizing treatment. This UIS could be a prognostic marker to predict the evolution towards CKD in cystinuria.

Neat plasma proteomics: getting the best out of the worst.

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.

Characterization of GEXP15 as a Potential Regulator of Protein Phosphatase 1 in Plasmodium falciparum.

The Protein Phosphatase type 1 catalytic subunit (PP1c) (PF3D7_1414400) operates in combination with various regulatory proteins to specifically direct and control its phosphatase activity. However, there is little information about this phosphatase and its regulators in the human malaria parasite, Plasmodium falciparum. To address this knowledge gap, we conducted a comprehensive investigation into the structural and functional characteristics of a conserved Plasmodium-specific regulator called Gametocyte EXported Protein 15, GEXP15 (PF3D7_1031600). Through in silico analysis, we identified three significant regions of interest in GEXP15: an N-terminal region housing a PP1-interacting RVxF motif, a conserved domain whose function is unknown, and a GYF-like domain that potentially facilitates specific protein-protein interactions. To further elucidate the role of GEXP15, we conducted in vitro interaction studies that demonstrated a direct interaction between GEXP15 and PP1 via the RVxF-binding motif. This interaction was found to enhance the phosphatase activity of PP1. Additionally, utilizing a transgenic GEXP15-tagged line and live microscopy, we observed high expression of GEXP15 in late asexual stages of the parasite, with localization predominantly in the nucleus. Immunoprecipitation assays followed by mass spectrometry analyses revealed the interaction of GEXP15 with ribosomal- and RNA-binding proteins. Furthermore, through pull-down analyses of recombinant functional domains of His-tagged GEXP15, we confirmed its binding to the ribosomal complex via the GYF domain. Collectively, our study sheds light on the PfGEXP15-PP1-ribosome interaction, which plays a crucial role in protein translation. These findings suggest that PfGEXP15 could serve as a potential target for the development of malaria drugs.

Proteomics of Plasma and Plasma-Treated Podocytes: Application to Focal and Segmental Glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is a severe form of idiopathic nephrotic syndrome (INS), a glomerulopathy of presumably immune origin that is attributed to extrarenal pathogenic circulating factors. The recurrence of FSGS (rFSGS) after transplant occurs in 30% to 50% of cases. The direct analysis of patient plasma proteome has scarcely been addressed to date, mainly due to the methodological difficulties associated with plasma complexity and dynamic range. In this study, first, we compared different methods of plasma preparation, second, we compared the plasma proteomes of rFSGS and controls using two preparation methods, and third, we analyzed the early proximal signaling events in podocytes subjected to patient plasma, through a combination of phosphoproteomics and lipid-raft proteomics (raftomics). By combining immunodepletion and high pH fractionation, we performed a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) obtained from healthy controls, non-INS patient controls, and rFSGS patients (n = 4). In both the soluble- and the EV-protein sets from the rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid-binding proteins, generally associated with lipoproteins, was found to be decreased in the soluble set from the rFSGS patients. In addition, three amino acid transporters involved in mTORC1 activation were found to be significantly increased in the EV from the rFSGS. Next, we incubated human podocytes for 30 min with 10% plasma from both groups of patients. The phosphoproteomics and raftomics of the podocytes revealed profound differences in the proteins involved in the mTOR pathway, in autophagy, and in cytoskeleton organization. We analyzed the correlation between the abundance of plasma and plasma-regulated podocyte proteins. The observed changes highlight some of the mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating-factor activity in podocytes.

Pancreatic islet cell stress induced by insulin-degrading enzyme deficiency promotes islet regeneration and protection from autoimmune diabetes.

Appropriate tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with highly variable demand for insulin synthesis. An efficient UPR ensures a sufficient beta cell mass and secretory output but can also affect beta cell resilience to autoimmune aggression. The factors regulating protein homeostasis in the face of metabolic and immune challenges are insufficiently understood. We examined beta cell adaptation to stress in mice deficient for insulin-degrading enzyme (IDE), a ubiquitous protease with high affinity for insulin and genetic association with type 2 diabetes. IDE deficiency induced a low-level UPR in both C57BL/6 and autoimmune non-obese diabetic (NOD) mice, associated with rapamycin-sensitive beta cell proliferation strongly enhanced by proteotoxic stress. Moreover, in NOD mice, IDE deficiency protected from spontaneous diabetes and triggered an additional independent pathway, conditional on the presence of islet inflammation but inhibited by proteotoxic stress, highlighted by strong upregulation of regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. Our findings establish a key role of IDE in islet cell protein homeostasis, identify a link between low-level UPR and proliferation, and reveal an UPR-independent anti-inflammatory islet cell response uncovered in the absence of IDE of potential interest in autoimmune diabetes.

Promitotic Action of Oenothera biennis on Senescent Human Dermal Fibroblasts.

Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of Oenothera biennis cell cultures (ObHEx) and we showed its skin anti-aging properties. The aim of this work is to assess its biological effect specifically on cell senescence. ObHEx action has been evaluated on normal human dermal fibroblasts subjected to stress-induced premature senescence (SIPS) through an ultra-deep proteomic analysis, leading to the most global senescence-associated proteome so far. Mass spectrometry data show that the treatment with ObHEx re-establishes levels of crucial mitotic proteins, strongly downregulated in senescent cells. To validate our proteomics findings, we proved that ObHEx can, in part, restore the activity of 'senescence-associated-ß-galactosidase', the most common hallmark of senescent cells. Furthermore, to assess if the upregulation of mitotic protein levels translates into a cell cycle re-entry, FACS experiments have been carried out, demonstrating a small but significative reactivation of senescent cell proliferation by ObHEx. In conclusion, the deep senescence-associated global proteome profiling published here provides a panel of hundreds of proteins deregulated by SIPS that can be used by the community to further understand senescence and the effect of new potential modulators. Moreover, proteomics analysis pointed to a specific promitotic effect of ObHEx on senescent cells. Thus, we suggest ObHEx as a powerful adjuvant against senescence associated with skin aging.

The Heterochromatin protein 1 is a regulator in RNA splicing precision deficient in ulcerative colitis.

Defects in RNA splicing have been linked to human disorders, but remain poorly explored in inflammatory bowel disease (IBD). Here, we report that expression of the chromatin and alternative splicing regulator HP1γ is reduced in ulcerative colitis (UC). Accordingly, HP1γ gene inactivation in the mouse gut epithelium triggers IBD-like traits, including inflammation and dysbiosis. In parallel, we find that its loss of function broadly increases splicing noise, favoring the usage of cryptic splice sites at numerous genes with functions in gut biology. This results in the production of progerin, a toxic splice variant of prelamin A mRNA, responsible for the Hutchinson-Gilford Progeria Syndrome of premature aging. Splicing noise is also extensively detected in UC patients in association with inflammation, with progerin transcripts accumulating in the colon mucosa. We propose that monitoring HP1γ activity and RNA splicing precision can help in the management of IBD and, more generally, of accelerated aging.

Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters.

Proteins interacting with CFTR and its mutants have been intensively studied using different experimental approaches. These studies provided information on the cellular processes leading to proper protein folding, routing to the plasma membrane, recycling, activation and degradation. Recently, new approaches have been developed based on the proximity labeling of protein partners or proteins in close vicinity and their subsequent identification by mass spectrometry. In this study, we evaluated TurboID- and APEX2-based proximity labeling of WT CFTR and compared the obtained data to those reported in databases. The CFTR-WT interactome was then compared to that of two CFTR (G551D and W1282X) mutants and the structurally unrelated potassium channel KCNK3. The two proximity labeling approaches identified both known and additional CFTR protein partners, including multiple SLC transporters. Proximity labeling approaches provided a more comprehensive picture of the CFTR interactome and improved our knowledge of the CFTR environment.

Plasmodium berghei leucine-rich repeat protein 1 downregulates protein phosphatase 1 activity and is required for efficient oocyst development.

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1-LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.

UNC45A deficiency causes microvillus inclusion disease-like phenotype by impairing myosin VB-dependent apical trafficking.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Extracellular Vesicles From LPS-Treated Macrophages Aggravate Smooth Muscle Cell Calcification by Propagating Inflammation and Oxidative Stress.

Background: Vascular calcification (VC) is a cardiovascular complication associated with a high mortality rate among patients with diseases such as atherosclerosis and chronic kidney disease. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete a heterogeneous population of extracellular vesicles (EVs). Recent studies have shown involvement of EVs in the inflammation and oxidative stress observed in VC. We aimed to decipher the role and mechanism of action of macrophage-derived EVs in the propagation of inflammation and oxidative stress on VSMCs during VC. Methods: The macrophage murine cell line RAW 264.7 treated with lipopolysaccharide (LPS-EK) was used as a cellular model for inflammatory and oxidative stress. EVs secreted by these macrophages were collected by ultracentrifugation and characterized by transmission electron microscopy, cryo-electron microscopy, nanoparticle tracking analysis, and the analysis of acetylcholinesterase activity, as well as that of CD9 and CD81 protein expression by western blotting. These EVs were added to a murine VSMC cell line (MOVAS-1) under calcifying conditions (4 mM Pi-7 or 14 days) and calcification assessed by the o-cresolphthalein calcium assay. EV protein content was analyzed in a proteomic study and EV cytokine content assessed using an MSD multiplex immunoassay. Results: LPS-EK significantly decreased macrophage EV biogenesis. A 24-h treatment of VSMCs with these EVs induced both inflammatory and oxidative responses. LPS-EK-treated macrophage-derived EVs were enriched for pro-inflammatory cytokines and CAD, PAI-1, and Saa3 proteins, three molecules involved in inflammation, oxidative stress, and VC. Under calcifying conditions, these EVs significantly increase the calcification of VSMCs by increasing osteogenic markers and decreasing contractile marker expression. Conclusion: Our results show that EVs derived from LPS-EK-treated-macrophages are able to induce pro-inflammatory and pro-oxidative responses in surrounding cells, such as VSMCs, thus aggravating the VC process.

Mapping PP1c and Its Inhibitor 2 Interactomes Reveals Conserved and Specific Networks in Asexual and Sexual Stages of Plasmodium.

Malaria parasites require multiple phosphorylation and dephosphorylation steps to drive signaling pathways for proper differentiation and transformation. Several protein phosphatases, including protein phosphatase 1 (PP1), one of the main dephosphorylation enzymes, have been shown to be indispensable for the Plasmodium life cycle. The catalytic subunit of PP1 (PP1c) participates in cellular processes via dynamic interactions with a vast number of binding partners that contribute to its diversity of action. In this study, we used Plasmodium berghei transgenic parasite strains stably expressing PP1c or its inhibitor 2 (I2) tagged with mCherry, combined with the mCherry affinity pulldown of proteins from asexual and sexual stages, followed by mass spectrometry analyses. Mapped proteins were used to identify interactomes and to cluster functionally related proteins. Our findings confirm previously known physical interactions of PP1c and reveal enrichment of common biological processes linked to cellular component assembly in both schizonts and gametocytes to biosynthetic processes/translation in schizonts and to protein transport exclusively in gametocytes. Further, our analysis of PP1c and I2 interactomes revealed that nuclear export mediator factor and peptidyl-prolyl cis-trans isomerase, suggested to be essential in P. falciparum, could be potential targets of the complex PP1c/I2 in both asexual and sexual stages. Our study emphasizes the adaptability of Plasmodium PP1 and provides a fundamental study of the protein interaction landscapes involved in a myriad of events in Plasmodium, suggesting why it is crucial to the parasite and a source for alternative therapeutic strategies.

BLI-MS: Combining biolayer interferometry and mass spectrometry.

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.

RESVEGA, a Nutraceutical Omega-3/Resveratrol Supplementation, Reduces Angiogenesis in a Preclinical Mouse Model of Choroidal Neovascularization.

Age-related macular degeneration (AMD) is an eye disease that is characterized by damage to the central part of the retina, the macula, and that affects millions of people worldwide. At an advanced stage, a blind spot grows in the center of vision, severely handicapping patients with this degenerative condition. Despite therapeutic advances thanks to the use of anti-VEGF, many resistance mechanisms have been found to accentuate the visual deficit. In the present study, we explored whether supplementation with Resvega®, a nutraceutical formulation composed of omega-3 fatty acids and resveratrol, a well-known polyphenol in grapes, was able to counteract laser-induced choroidal neovascularization (CNV) in mice. We highlight that Resvega® significantly reduced CNV in mice compared with supplementations containing omega-3 or resveratrol alone. Moreover, a proteomic approach confirmed that Resvega® could counteract the progression of AMD through a pleiotropic effect targeting key regulators of neoangiogenesis in retina cells in vivo. These events were associated with an accumulation of resveratrol metabolites within the retina. Therefore, a supplementation of omega-3/resveratrol could improve the management or slow the progression of AMD in patients with this condition.

STING protects breast cancer cells from intrinsic and genotoxic-induced DNA instability via a non-canonical, cell-autonomous pathway.

STING (Stimulator of Interferon Genes) is an endoplasmic reticulum-anchored adaptor of the innate immunity best known to trigger pro-inflammatory cytokine expression in response to pathogen infection. In cancer, this canonical pathway can be activated by intrinsic or drug-induced genomic instability, potentiating antitumor immune responses. Here we report that STING downregulation decreases cell survival and increases sensitivity to genotoxic treatment in a panel of breast cancer cell lines in a cell-autonomous manner. STING silencing impaired DNA Damage Response (53BP1) foci formation and increased DNA break accumulation. These newly identified properties were found to be independent of STING partner cGAS and of its canonical pro-inflammatory pathway. STING was shown to partially localize at the inner nuclear membrane in a variety of breast cancer cell models and clinical tumor samples. Interactomics analysis of nuclear STING identified several proteins of the DNA Damage Response, including the three proteins of the DNA-PK complex, further supporting a role of STING in the regulation of genomic stability. In breast and ovarian cancer patients that received adjuvant chemotherapy, high STING expression is associated with increased risk of relapse. In summary, this study highlights an alternative, non-canonical tumor-promoting role of STING that opposes its well-documented function in tumor immunosurveillance.

Polo-like kinase 1 (Plk1) regulates DNA replication origin firing and interacts with Rif1 in Xenopus.

The activation of eukaryotic DNA replication origins needs to be strictly controlled at multiple steps in order to faithfully duplicate the genome and to maintain its stability. How the checkpoint recovery and adaptation protein Polo-like kinase 1 (Plk1) regulates the firing of replication origins during non-challenged S phase remained an open question. Using DNA fiber analysis, we show that immunodepletion of Plk1 in the Xenopus in vitro system decreases replication fork density and initiation frequency. Numerical analyses suggest that Plk1 reduces the overall probability and synchrony of origin firing. We used quantitative chromatin proteomics and co-immunoprecipitations to demonstrate that Plk1 interacts with firing factors MTBP/Treslin/TopBP1 as well as with Rif1, a known regulator of replication timing. Phosphopeptide analysis by LC/MS/MS shows that the C-terminal domain of Rif1, which is necessary for its repressive action on origins through protein phosphatase 1 (PP1), can be phosphorylated in vitro by Plk1 on S2058 in its PP1 binding site. The phosphomimetic S2058D mutant interrupts the Rif1-PP1 interaction and modulates DNA replication. Collectively, our study provides molecular insights into how Plk1 regulates the spatio-temporal replication program and suggests that Plk1 controls origin activation at the level of large chromatin domains in vertebrates.

The pentose phosphate pathway constitutes a major metabolic hub in pathogenic Francisella.

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.

Identification and Relative Quantification of hFSH Glycoforms in Women's Sera via MS-PRM-Based Approach.

Follicle-stimulating hormone (FSH) is a glycohormone synthesized by adenohypophysis, and it stimulates ovulation in women and spermatogenesis in men by binding to its receptor (FSHR). FSHR is involved in several mechanisms to transduce intracellular signals in response to the FSH stimulus. Exogenous FSH is currently used in the clinic for ovarian hyperstimulation during in vitro fertilization in women, and for treatment of infertility caused by gonadotropin deficiency in men. The glycosylation of FSH strongly affects the binding affinity to its receptor, hence significantly influencing the biological activity of the hormone. Therefore, the accurate measurement and characterization of serum hFSH glycoforms will contribute to elucidating the complex mechanism of action by which different glycoforms elicit distinct biological activity. Nowadays ELISA is the official method with which to monitor serum hFSH, but the test is unable to distinguish between the different FSH glycovariants and is therefore unsuitable to study the biological activity of this hormone. This study presents a preliminary alternative strategy for identifying and quantifying serum hFSH glycoforms based on immunopurification assay and mass spectrometry (MS), and parallel reaction monitoring (PRM) analysis. In this study, we provide an MS-PRM data acquisition method for hFSH glycopeptides identification with high specificity and their quantification by extracting the chromatographic traces of selected fragments of glycopeptides. Once set up for all its features, the proposed method could be transferred to the clinic to improve fertility treatments and follow-ups in men and women.