Diel Cycle Proteomics: Illuminating Molecular Dynamics in Purple Bacteria for Optimized Biotechnological Applications

Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily variations. While ubiquitous in Eukaryotes, circadian clocks remain exclusively characterized in Cyanobacteria among Prokaryotes. These rhythms are regulated by a core oscillator, which is controlled by a cluster of three genes: kaiA, kaiB, and kaiC. Interestingly, recent studies revealed rhythmic activities, potentially tied to a circadian clock, in other Prokaryotes, including purple bacteria such as Rhodospirillum rubrum, known for its applications in fuel and plastic bioproduction. However, the pivotal question of how light and dark cycles influence protein dynamics and the expression of putative circadian clock genes remains unexplored in purple non-sulfur bacteria. Unraveling the regulation of these molecular clocks holds the key to unlocking optimal conditions for harnessing the biotechnological potential of R. rubrum. Understanding how its proteome responds to different light regimes—whether under continuous light or alternating light and dark cycles—could pave the way for precisely fine-tuning bioproduction processes. Here, we report for the first time the expressed proteome of R. rubrum grown under continuous light versus light and dark cycle conditions using a shotgun proteomic analysis. In addition, we measured the impact of light regimes on the expression of four putative circadian clock genes (kaiB1, kaiB2, kaiC1, kaiC2) at the transcriptional and translational levels using RT-qPCR and targeted proteomic (MRM-MS), respectively. The data revealed significant effects of light conditions on the overall differential regulation of the proteome, particularly during the early growth stages. Notably, several proteins were found to be differentially regulated during the light or dark period, thus impacting crucial biological processes such as energy conversion pathways and the general stress response. Furthermore, our study unveiled distinct regulation of the four kai genes at both the mRNA and protein levels in response to varying light conditions. Deciphering the impact of the diel cycle on purple bacteria not only enhances our understanding of their ecology but also holds promise for optimizing their applications in biotechnology, providing valuable insights into the origin and evolution of prokaryotic clock mechanisms

Proteomic differences in seminal fluid of social insects whose sperm differ in heat tolerance.

peer reviewedIn the coming years, climate change is likely to increase the frequency and intensity of heatwaves. In many organisms, heat stress provokes physiological perturbations and can lead to decreased male fertility. Bumblebees are endo-heterothermic but display interspecific differences in thermotolerance that could have conservation implications. For the species of concern Bombus magnus, exposure to high temperatures can severely reduce sperm quality and, consequently, reproductive success. Such is not the case for B. terrestris, a ubiquitous species. To decipher the mechanisms at play, we characterized the seminal fluid proteomes of the two species. We quantified 1121 proteins, of which 522 were differentially expressed between B. terrestris and B. magnus. Several proteins with protective functions, such as proteases, antioxidant proteins and various heat-shock proteins, were present at higher levels in B. terrestris than in B. magnus under both control and heat-stress conditions. The same was true for proteins involved in cellular homeostasis, immunity, lipid/sugar metabolism and thermotolerance. Furthermore, proteins involved in the capture and elimination of reactive oxygen species also occurred at much high levels in B. terrestris. Overall, these results clearly indicate differences in the seminal proteome of the more thermotolerant B. terrestris versus B. magnus. The differences may contribute to explaining interspecific differences in sperm survival

Mass spectrometry analysis of saponins

peer reviewedSaponins are amphiphilic molecules of pharmaceutical interest and most of their biological activities (i.e. cytotoxic, hemolytic, fungicide...) are associated to their membranolytic properties. These molecules are secondary metabolites present in numerous plants and in some marine animals, such as sea cucumbers and starfishes. Structurally, all saponins correspond to the combination of a hydrophilic glycan, consisting of sugar chain(s), linked to a hydrophobic triterpenoidic or steroidic aglycone, named the sapogenin. Saponins present a high structural diversity and their structural characterization remains extremely challenging. Ideally, saponin structures are best established using nuclear magnetic resonance experiments conducted on isolated molecules. However, the extreme structural diversity of saponins makes them challenging from a structural analysis point of view since, most of the time, saponin extracts consist in a huge number of congeners presenting only subtle structural differences. In the present review, we wish to offer an overview of the literature related to the development of mass spectrometry for the study of saponins. This review will demonstrate that most of the past and current mass spectrometry methods, including electron, electrospray and MALDI ionizations, gas/liquid chromatography coupled to (tandem) mass spectrometry, collision-induced dissociation including MS3 experiments, multiple reaction monitoring based quantification, ion mobility experiments... have been used for saponin investigations with great success on enriched extracts but also directly on tissues using imaging methods

Identification and quantification of spinochromes in body compartments of Echinometra mathaei's coloured types

peer reviewe

Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process

peer reviewe

Ion Mobility Mass Spectrometry of Saponin Ions

peer reviewe

Triterpenoids in Echinoderms: Fundamental Differences in Diversity and Biosynthetic Pathways

Echinoderms form a remarkable phylum of marine invertebrates that present specific chemical signatures unique in the animal kingdom. It is particularly the case for essential triterpenoids that evolved separately in each of the five echinoderm classes. Indeed, while most animals have ∆5-sterols, sea cucumbers (Holothuroidea) and sea stars (Asteroidea) also possess ∆7 and ∆9(11)-sterols, a characteristic not shared with brittle stars (Ophiuroidea), sea urchins (Echinoidea), and crinoids (Crinoidea). These particular ∆7 and ∆9(11) sterols emerged as a self-protection against membranolytic saponins that only sea cucumbers and sea stars produce as a defense mechanism. The diversity of saponins is large; several hundred molecules have been described in the two classes of these saponins (i.e., triterpenoid or steroid saponins). This review aims to highlight the diversity of triterpenoids in echinoderms by focusing on sterols and triterpenoid glycosides, but more importantly to provide an updated view of the biosynthesis of these molecules in echinoderms

Développement d'une trousse de dosage d'organokines par spectrométrie de masse

Lors d'une prise en charge thérapeutique, l'identification de marqueurs biologiques permettant un meilleur diagnostic ainsi qu'une caractérisation évolutive du patient est essentielle. A cet égard, les organokines occupent une place importante car elles sont considérées comme des médiateurs moléculaires de la communication (cross-talk) inter-organes. Nous développons actuellement une méthode de quantification multiplexe de ces organokines. Cette recherche s'inscrit dans une des thématiques phares de l'Institut de Recherche Santé de l'UMONS et résulte d'une collaboration entre 6 services de l'Institut dans le domaine de la biomédecine intégrative. L'identification d'altérations du cross-talk inter-organes vise au développement futur de nouvelles stratégies thérapeutiques dans différents contextes pathologiques. Les organokines sont produites à partir de divers organes tels le tissu adipeux (adipokines), les muscles striés squelettiques (myokines), le foie (hépatokines), ou encore le cerveau (neurokines). Chacune de ces molécules est relarguée dans la circulation sanguine et agit ensuite à distance au niveau d'autres tissus et organes, jouant un rôle essentiel dans la coordination et le maintien de l'homéostasie, notamment métabolique. Il est aujourd'hui reconnu qu'une production ainsi qu'une libération excessive et inappropriée de ces organokines participe au développement et à la progression de pathologies comme l'obésité, le diabète de type 2 et les maladies cardiovasculaires. Actuellement, les organokines sont dosées individuellement par l'utilisation de tests ELISA ou RIA. Ces approches, non seulement coûteuses, n'existent pas pour toutes les organokines d'intérêt. Dans ce contexte, la spectrométrie de masse, approche très sensible, permet la quantification simultanée de nombreuses protéines dans différentes matrices biologiques dont le sérum ou le plasma. Ce projet implique une approche translationnelle directement en lien avec la clinique. Cette approche permettra d'acquérir une vision globale et intégrée des mécanismes de communication inter-organes impliqués dans les pathologies étudiées et de développer ainsi des stratégies thérapeutiques innovantes

Impact des organokines dans l'insuffisance rénale induite par l'obésité

The growing increase of obesity intensifies the incidence of chronic kidney disease (CKD) across the world. We previously showed that AMP-activated protein kinase (AMPK) plays a critical role in regulating the chronic cellular response to lipid excess. Indeed, we established that high-fat diet (HFD)-induced kidney disease is characterized by renal hypertrophy, increased albuminuria and elevated markers of renal fibrosis and inflammation while these HFD-induced markers were reversed by pharmacological AMPK activation. In addition, we also showed that HFD alters lipid metabolism, leading to lipid accumulation in the kidney. While these features were highlighted in male mice, we recently demonstrated that female mice are protected from disturbed renal lipid metabolism. Indeed, lipidomic analysis revealed a significant increase in ceramide and phospholipids species in HFD-fed mice but not in HFD-fed female. Interestingly, these results were associated to a sustained plasma adiponectin level and renal AMPK activity (Juszczak et al., 2023). Therefore, to better understand the organ crosstalk during obesity-induced kidney disease, different organokines will be analyzed. Organokines like myokines, adipokines, hepatokines are peptides released in the blood by different organs (muscle, adipose tissue or liver respectively). Organokines may impact entire body metabolism and by extension renal function. In an obesity context, adipokine production variates leading to metabolic perturbation to other organs. Consequently, these latter will have an alteration of their organokine production leading to global organ cross talk disturbance. Here, we will characterize the organokines produced in male and female lean and obese mice and investigate their impact on the AMPK activity and renal lipotoxicity in primary proximal tubular epithelial cells. To do so, blood organokine levels are studied thank to Multiple Reaction Monitoring - Mass Spectrometry (MRM-MS) (MS-QUANTA, Bioprofiling platform, UMONS) and correlations are carried out with metabolic and renal function readouts. Up to now, adiponectin, leptin and adipsin are analyzed in MRM MS. First of all, serum organokine levels were analyzed in lean female and male mice with MRM-MS. Serum adiponectin level was significantly higher in female compared to male (Figure 1A). However, leptin and adipsin levels did not show any difference (Figures 1B and 1C-1). Thereafter, mice were fed with HFD and low-fat diet (LFD) for 20 weeks. Only adipsin level was reduced in obese male mice compared to lean male (Figure 1C-2). Currently, more analyzes are performing in HFD fed female mice, on kidney injury markers and on renal lipid metabolism to perform correlation with organokine level. Our principal objective is to develop organokine dosage method based on MRM-MS which is cheaper and more sensitive than antibody-based methods. This approach will be further developed in clinical studies in order to better understand organ cross talk mechanism notably in obesity induced CKD. This method aims to determine other potential biomarkers and to help therapeutic strategy