Molecular mechanisms underlying congenital neutropenia in patients with HAX1 deficiency are not clear at the moment. HAX1 deficiency was shown to result in the arrest of neutrophil differentiation. Our studies of the effect of HAX1 deficiency on the proteomic and metabolic profiles of promyelocytic cells have led to the conclusion, supported by specific tests, that fatty acid metabolism is affected in HAX1 KO cells. The lipid droplet content is increased in HAX1 KO cells, pointing to the accumulation of fatty acids that are not metabolized. Studies of autophagosome function in HAX1 WT and KO cells revealed that lipid droplet autophagy is defective at the stage of fusion with the lysosome. Autophagy-dependent generation of free fatty acids is critical for neutrophil differentiation, so HAX1 deficiency that affects normal autophagy of lipids in promyeloblasts should explain differentiation arrest. Moreover, we have demonstrated that HAX1-deficient cells are also compromised in fatty acid uptake.

Sugar starvation during seed germination requires coordinated regulation of reserve mobilization, redox homeostasis, and intracellular recycling. In lupin seeds, asparagine is a major nitrogen-rich metabolite, but its role in starvation-induced autophagy and redox regulation remains unclear. Here, isolated embryonic axes of white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet) were cultured in vitro under sucrose-fed or sugar-starved conditions, with or without asparagine supplementation. Using transcriptomic, proteomic, immunoblot, enzymatic, antioxidant activity, and confocal microscopy analyses, we show that sugar starvation induced redox- and autophagy-related reprogramming, including changes in reactive oxygen species (ROS)-related proteins, catalase accumulation, autophagy-related (ATG) gene expression, vacuolar hydrolase-related responses, and proteolytic activity. Peroxisome-associated components, including glycolate oxidase, acyl-CoA oxidase, and catalase, were strongly affected, indicating dynamic remodeling of peroxisome-related metabolism during starvation. Asparagine modified this response by increasing antioxidant capacity and catalase accumulation under sugar starvation, while reducing detectable autophagosome number, many ATG and vacuolar hydrolase transcripts, and proteolytic activity. Together with previous evidence for asparagine-induced accumulation of autophagic bodies in vacuoles, these results are consistent with asparagine-dependent modulation of several autophagy-related processes rather than with an effect restricted to a single autophagic step. White and Andean lupin shared the same general regulatory framework but differed in response intensity. Thus, asparagine links nitrogen status with redox stabilization, vacuolar catabolism, and autophagy-related dynamics in sugar-starved lupin embryonic axes.

Clathrin-mediated endocytosis (CME) internalizes cell-surface receptors via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to these sites by the adaptor protein complex AP2. AP2 cycles between a closed cytoplasmic conformation and an open membrane-bound state, and efficient CME requires both conformations and their dynamic interconversion. The mechanisms regulating these conformational changes, which include post-translational modifications of the AP2, remain incompletely understood. Here, we report that, in human cell lines, p70S6 kinase phosphorylates the μ2 subunit of the AP2 and that the phosphorylation of serine 45 (S45) depends on p70S6K activity. Loss of S45-μ2 phosphorylation results in decreased internalization of canonical CME cargo, such as transferrin and PDGF receptors. In Caenorhabditis elegans, lack of S45-μ2 phosphorylation produces directionally similar but markedly weaker phenotypes than AP2 loss of function. Live imaging and computational dynamic modelling suggest that S45-μ2 phosphorylation has an impact on the conformational changes of the AP2 complex. These findings identify a p70S6K-dependent mechanism that modulates AP2 function and further strengthen the importance of post-translational regulation in controlling CME.

Clathrin-mediated endocytosis (CME) is a process in which ligands and their corresponding receptors at the cell surface are internalized via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to the clathrin-coated pit by the adaptor protein complex AP2. AP2 resides in the cytoplasm in the closed conformation and opens upon interacting with the plasma membrane. Effective CME requires both pools of AP2, open and closed, and effective transition between these states but the mechanisms regulating this transition are only partially understood. Here, we report that serine 45 (S45) of the µ2 subunit of the AP2 complex is phosphorylated in a p70S6 kinase-dependent manner in HeLa cells both with hyperactivated mTOR signaling and under basal conditions. We demonstrate that the loss of S45-µ2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. In Caenorhabditis elegans, the absence of S45-µ2 phosphorylation produces the dumpy phenotype characteristic of the loss of function of AP2. Our live imaging experiments further suggest that these phenotypes might arise because S45-μ2 phosphorylation is needed for conformational changes of the AP2 complex. These findings uncover a mechanism central to CME control and extend our knowledge on the role of post-translational modifications of AP2 components in regulating the function of this complex. ### Competing Interest Statement The authors have declared no competing interest.

Racehorses undergo profound physiological changes with training and competition, but current biomarkers inadequately capture the complex molecular dynamics of exercise. This study aimed to identify novel plasma biomarkers of training adaptation and peak load using high-throughput proteomics. We hypothesised that systematic training and racing induce distinct plasma proteomic signatures, enabling the discovery of candidate biomarkers linked to training status, oxidative stress, inflammation and metabolic remodelling. In vivo longitudinal study. Forty-nine Arabian and Thoroughbred racehorses underwent standardised high-intensity training. Plasma samples were collected at rest, immediately post-exercise and after recovery during three phases: initial training (T1), mid-season conditioning (T2) and race-phase (R). In total, 314 samples were analysed using tandem mass tags based quantitative proteomics and Orbitrap mass spectrometry. Protein abundance changes were assessed with multiple-testing correction ( q  < 0.05), and pathway enrichment was performed using STRING and ShinyGO. Proteomic responses differed by phase. T1 showed broad activation of inflammatory (S100A8/A9), antioxidant (superoxide dismutase 1, catalase) and metabolic proteins (glucose-6-phosphate dehydrogenase, phosphoglycerate kinase 1). T2 displayed a more refined profile with remodelling and redox regulators (decorin, thymosin β4, glutathione S-transferase). Racing elicited the strongest response, with over 100 up-regulated proteins linked to energy metabolism, oxidative defense and cytoskeletal adaptation. Several proteins: including S100A8, thymosin β4, prothymosin-α, cofilin-1 and lipocalins, were consistently modulated across phases, highlighting their biomarker potential. Breed imbalance and incomplete follow-up sampling may affect generalisability. Validation in larger, diverse cohorts with targeted assays is required. This study identifies a panel of promising plasma proteins as candidate biomarkers of exercise adaptation and overload in racehorses. These findings may support improved monitoring of performance, training load and early detection of overtraining in equine athletes.

BACKGROUND:Racehorses undergo profound physiological changes with training and competition, but current biomarkers inadequately capture the complex molecular dynamics of exercise. This study aimed to identify novel plasma biomarkers of training adaptation and peak load using high-throughput proteomics. OBJECTIVES:We hypothesised that systematic training and racing induce distinct plasma proteomic signatures, enabling the discovery of candidate biomarkers linked to training status, oxidative stress, inflammation and metabolic remodelling. STUDY DESIGN:In vivo longitudinal study. METHODS:Forty-nine Arabian and Thoroughbred racehorses underwent standardised high-intensity training. Plasma samples were collected at rest, immediately post-exercise and after recovery during three phases: initial training (T1), mid-season conditioning (T2) and race-phase (R). In total, 314 samples were analysed using tandem mass tags based quantitative proteomics and Orbitrap mass spectrometry. Protein abundance changes were assessed with multiple-testing correction (q < 0.05), and pathway enrichment was performed using STRING and ShinyGO. RESULTS:Proteomic responses differed by phase. T1 showed broad activation of inflammatory (S100A8/A9), antioxidant (superoxide dismutase 1, catalase) and metabolic proteins (glucose-6-phosphate dehydrogenase, phosphoglycerate kinase 1). T2 displayed a more refined profile with remodelling and redox regulators (decorin, thymosin β4, glutathione S-transferase). Racing elicited the strongest response, with over 100 up-regulated proteins linked to energy metabolism, oxidative defense and cytoskeletal adaptation. Several proteins: including S100A8, thymosin β4, prothymosin-α, cofilin-1 and lipocalins, were consistently modulated across phases, highlighting their biomarker potential. MAIN LIMITATIONS:Breed imbalance and incomplete follow-up sampling may affect generalisability. Validation in larger, diverse cohorts with targeted assays is required. CONCLUSIONS:This study identifies a panel of promising plasma proteins as candidate biomarkers of exercise adaptation and overload in racehorses. These findings may support improved monitoring of performance, training load and early detection of overtraining in equine athletes.

There is a huge disparity between the number of bacterial and yeast probiotics in favor of the former. The latest reports indicate that extracellular vehicles (EVs) play a significant role in probiotic mechanisms. In the present work, we compared the probiotic properties of Saccharomyces cerevisiae strains (WUT3 and WUT151), which have never been previously characterized in this context, with commercial probiotic yeast-Saccharomyces cerevisiae var. boulardii CNCM-745. Notably, WUT3 and WUT151 reacted more mildly to the unfavorable simulated environment of saliva, stomach, small, and large intestines. As a result, we confirmed that WUT3 and WUT151 were superior to S. boulardii in terms of probiotic properties. Then, we performed a complex analysis of their EVs, isolated by a multistep filtration process. The nanoparticle tracing analysis showed no significant difference in the diameter of the vesicles between the strains. MTT studies confirmed that EVs are not toxic against normal human colorectal cell lines CCD-18 Co and CCD 841 CoN. However, toxicity was observed against the HT-29 cancer line. By staining EVs with Nile Red, we successfully visualized EVs-cell interactions. Finally, we explored the profile of proteins transported with the EVs, identifying a significant overrepresentation of extracellular proteins. Based on comparison with other proteomic data, we selected marker proteins for S. cerevisiae EVs. This knowledge will be helpful for further studies on tracking the transfer of the protein cargo of yeast EVs to human cells using, for instance, specific antibodies to these marker proteins.

Zika virus is a teratogenic pathogen belonging to the Flaviviridae family. It possesses the ability to penetrate the placenta and affect the brain development of a fetus, resulting in microcephaly and functional impairments. Mechanisms of this neurotoxicity are still unclear, but capsid proteins of Zika and related viruses are known to exert apoptotic effect in different types of cells, including neurons. To explore the pathways affected by the presence of ZIKV-C, we have performed MS-based interactomic experiment in human neural stem cells and managed to identify 149 putative interactors. Our results indicate that the nucleus (especially the nucleolus) and the mitochondria are the main sites of interaction of protein C with host proteins. A number of the proteins we identified have significant links to diseases of the nervous system, including neurodevelopmental diseases. Furthermore, for the first time, we have identified MAM-domain containing glycosylphosphatidylinositol anchor protein 1, T-complex protein 1 subunit beta, lysine-tRNA ligase, calumenin as particularly abundant ZIKV protein C interactors. Data are available via ProteomeXchange with identifier PXD064412. ### Competing Interest Statement The authors have declared no competing interest. * ZIKV : Zika virus WNV : West Nile virus JEV : Japanese encephalitis virus NSCs : neural stem cells NPCs : neuroprogenitor cells ZIKV-C : protein C from Zika virus WNV-C : protein C from West Nile virus JEV-C : protein C from Japanese encephalitis viruses FDR : False Discovery Rate PPI : protein-protein interaction SVZ : subventricular zone CALU : calumenin KARS : lysine-tRNA ligase Polish National Science Centre, 2018/29/B/NZ2/01752

Violacein is a natural indole-derived purple pigment of microbial origin that has attracted attention for its remarkable biological properties. Due to its poor solubility in aqueous media, most studies of this pigment use extracts of the compound obtained with common solvents. Violacein is also transported in bacterial extracellular vesicles (EVs) and transferred via this type of carrier remains stable in an aqueous environment. This paper is the first to present an in-depth study of Janthinobacterium lividum EVs as violacein carriers. J. lividum EVs were studied for their contribution to violacein translocation, size, morphology and protein composition. The production of violacein encapsulated in EVs was more efficient than the intracellular production of this compound. The average size of the violacein-containing EVs was 124.07 ± 3.74 nm. Liquid chromatography-tandem mass spectrometry analysis (LC–MS/MS) revealed 932 proteins common to three independent EVs isolations. The high proportion of proteins with intracellular localisation, which are involved in many fundamental cellular processes, suggests that J. lividum EVs could be generated in a cell lysis model, additionally stimulated by violacein production. Using human keratinocytes and melanoma cell lines, it was confirmed that J. lividum EVs are able to react with and deliver their cargo to mammalian cells. The EVs-delivered violacein was shown to retain its activity against melanoma cells, and the dose and timing of treatment can be selected to target only cancer cells. The characterisation of J. lividum EVs, described in the following paper, represents a milestone for their future potential anticancer application. • This report focuses on the investigation of Janthinobacterium lividum EVs as a new delivery vehicle for violacein, a compound with a previously demonstrated broad spectrum of activity. • EVs were characterised for size, morphology and protein composition. • Studies on human keratinocytes and a melanoma cell model confirmed that the activity of violacein applied in the encapsulated form of EVs is similar to that of its organic solvent extract, but their production is much more environmentally friendly.

Background/Objectives: The pathophysiological background of the processes activated by physical activity in patients with heart failure (HF) is not fully understood. Proteomic studies can help to preliminarily identify new protein markers for unknown or poorly defined physiological processes. We aimed to analyse the changes in the plasma proteomic profile of HF patients after a cardiopulmonary exercise test (CPET) to define pathways involved in the response to exercise. Methods: The study prospectively enrolled 20 male patients with advanced HF (aged 53.3 ± 8.3 years). Blood samples were taken from the patients before and immediately after the CPET to obtain plasma proteomic profiles. Two-sample t-tests (paired or non-paired) were performed with and without false discovery rate (FDR) correction for multiple testing. Enrichment analysis was performed to associate biological processes and pathways with the study results. Results: A total of 968 plasma proteins were identified, of which 722 underwent further statistical analysis. Of these, 236 proteins showed differential expression when comparing all plasma samples collected before and after CPT (p < 0.05), and for 86 of these the difference remained statistically significant after FDR correction. Proteins whose expression changed after exercise are mostly involved in immune response and inflammatory processes, coagulation, cell adhesion, regulation of cellular response to stimulus and regulation of programmed cell death. There were no differences in resting proteomics according to HF etiology (ischemic vs. non-ischemic). Conclusions: Changes in the proteomic profile revealed a complexity of exercise-induced processes in patients with HF, suggesting that few major physiological pathways are involved. Further studies focusing on specific pathways are needed.

Under nutrient deficiency or starvation conditions, the mobilization of storage compounds during seed germination is enhanced to primarily supply respiratory substrates and hence increase the potential of cell survival. Nevertheless, we found that, under sugar starvation conditions in isolated embryonic axes of white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet) cultured in vitro for 96 h, the disruption of lipid breakdown occurs, as was reflected in the higher lipid content in the sugar-starved (-S) than in the sucrose-fed (+S) axes. We postulate that pexophagy (autophagic degradation of the peroxisome-a key organelle in lipid catabolism) is one of the reasons for the disruption in lipid breakdown under starvation conditions. Evidence of pexophagy can be: (i) the higher transcript level of genes encoding proteins of pexophagy machinery, and (ii) the lower content of the peroxisome marker Pex14p and its increase caused by an autophagy inhibitor (concanamycin A) in -S axes in comparison to the +S axes. Additionally, based on ultrastructure observation, we documented that, under sugar starvation conditions lipophagy (autophagic degradation of whole lipid droplets) may also occur but this type of selective autophagy seems to be restricted under starvation conditions. Our results also show that autophagy occurs at the very early stages of plant growth and development, including the cells of embryonic seed organs, and allows cell survival under starvation conditions.

Extracellular vesicles (EVs) are nanoparticles containing various bioactive cargos—e.g., proteins, RNAs, and lipids—that are released into the environment by all cell types. They are involved in, amongst other functions, intercellular communication. This article presents studies on EVs produced by the probiotic yeast Saccharomyces boulardii CNCM I-745. The size distribution and concentration of EVs in the liquid culture of yeast were estimated. Moreover, the vesicles of S. boulardii were tested for their cytotoxicity against three model human intestinal cell lines. This study did not show any significant negative effect of yeast EVs on these cells under tested conditions. In addition, EVs of S. boulardii were verified for their ability to internalize in vitro with human cells and transfer their cargo. The yeast vesicles were loaded with doxorubicin, an anticancer agent, and added to the cellular cultures. Subsequently, microscopic observations revealed that these EVs transferred the compound to human intestinal cell lines. A cytotoxicity test confirmed the activity of the transferred doxorubicin. Detailed information about the proteins present in EVs might be important in terms of exploring yeast EVs as carriers of active molecules. Thus, proteomic analysis of the EV content was also conducted within the present study, and it allowed the identification of 541 proteins after matching them to the Saccharomyces Genome Database (SGD). Altogether, this study provides strong evidence that the EVs of the probiotic CNCM I-745 strain could be considered a drug delivery system.

Abstract The disruption of the sumoylation pathway affects processes controlled by the two phototropins (phots) of Arabidopsis thaliana, phot1 and phot2. Phots, plant UVA/blue light photoreceptors, regulate growth responses and fast movements aimed at optimizing photosynthesis, such as phototropism, chloroplast relocations and stomatal opening. Sumoylation is a posttranslational modification, consisting of the addition of a SUMO (SMALL UBIQUITIN-RELATED MODIFIER) protein to a lysine residue in the target protein. In addition to affecting the stability of proteins, it regulates their activity, interactions and subcellular localization. We examined physiological responses controlled by phots, phototropism and chloroplast movements, in sumoylation pathway mutants. Chloroplast accumulation in response to both continuous and pulse light was enhanced in the E3 ligase siz1 mutant, in a manner dependent on phot2. A significant decrease in phot2 protein abundance was observed in this mutant after blue light treatment both in seedlings and mature leaves. Using plant transient expression and yeast two-hybrid assays, we found that phots interacted with SUMO proteins mainly through their N-terminal parts, which contain the photosensory LOV domains. The covalent modification in phots by SUMO was verified using an Arabidopsis sumoylation system reconstituted in bacteria followed by the mass spectrometry analysis. Lys 297 was identified as the main target of SUMO3 in the phot2 molecule. Finally, sumoylation of phot2 was detected in Arabidopsis mature leaves upon light or heat stress treatment.

The slime mold Dictyostelium discoideum's life cycle includes different unicellular and multicellular stages that provide a convenient model for research concerning intracellular and intercellular mechanisms influencing mitochondria's structure and function. We aim to determine the differences between the mitochondria isolated from the slime mold regarding its early developmental stages induced by starvation, namely the unicellular (U), aggregation (A) and streams (S) stages, at the bioenergetic and proteome levels. We measured the oxygen consumption of intact cells using the Clarke electrode and observed a distinct decrease in mitochondrial coupling capacity for stage S cells and a decrease in mitochondrial coupling efficiency for stage A and S cells. We also found changes in spare respiratory capacity. We performed a wide comparative proteomic study. During the transition from the unicellular stage to the multicellular stage, important proteomic differences occurred in stages A and S relating to the proteins of the main mitochondrial functional groups, showing characteristic tendencies that could be associated with their ongoing adaptation to starvation following cell reprogramming during the switch to gluconeogenesis. We suggest that the main mitochondrial processes are downregulated during the early developmental stages, although this needs to be verified by extending analogous studies to the next slime mold life cycle stages.

Motile cilia are ultrastructurally complex cell organelles with the ability to actively move. The highly conserved central apparatus of motile 9 × 2 + 2 cilia is composed of two microtubules and several large microtubule-bound projections, including the C1b/C1f supercomplex. The composition and function of C1b/C1f subunits has only recently started to emerge. We show that in the model ciliate Tetrahymena thermophila, C1b/C1f contains several evolutionarily conserved proteins: Spef2A, Cfap69, Cfap246/LRGUK, Adgb/androglobin, and a ciliate-specific protein Tt170/TTHERM_00205170. Deletion of genes encoding either Spef2A or Cfap69 led to a loss of the entire C1b projection and resulted in an abnormal vortex motion of cilia. Loss of either Cfap246 or Adgb caused only minor alterations in ciliary motility. Comparative analyses of wild-type and C1b-deficient mutant ciliomes revealed that the levels of subunits forming the adjacent C2b projection but not C1d projection are greatly reduced, indicating that C1b stabilizes C2b. Moreover, the levels of several IFT and BBS proteins, HSP70, and enzymes that catalyze the final steps of the glycolytic pathway: enolase ENO1 and pyruvate kinase PYK1, are also reduced in the C1b-less mutants.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. It affects many organs. Lewy bodies—a histopathological “hallmark” of PD—are detected in about 75% of PD submandibular gland samples. We hypothesize that saliva can be a source of biomarkers of PD. The aim of the study was to evaluate and compare the salivary proteome of PD patients and healthy controls (HC). Salivary samples from 39 subjects (24 PD patients, mean age 61.6 ± 8.2; 15 HC, mean age 60.9 ± 6.7) were collected. Saliva was collected using RNA-Pro-Sal kits. Label-free LC-MS/MS mass spectrometry was performed to characterize the proteome of the saliva. IPA analysis of upstream inhibitors was performed. A total of 530 proteins and peptides were identified. We observed lower concentrations of S100-A16, ARP2/3, and VPS4B in PD group when compared to HC. We conclude that the salivary proteome composition of PD patients is different than that of healthy controls. We observed a lower concentration of proteins involved in inflammatory processes, exosome formation, and adipose tissue formation. The variability of expression of proteins between the two groups needs to be considered.

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrantm RNAs comprising a premature translation termination codon. The adenosine triphosphate (ATP)-dependent RNA helicase up-frameshift 1 (UPF1) is a major NMD factor in all studied organisms; however, the complexity of this mechanism has not been fully characterized in plants. To identify plant NMD factors, we analyzed UPF1-interacting proteins using tandem affinity purification coupled to mass spectrometry.Canonical members of the NMD pathway were found along with numerous NMD candidate factors, including conserved DEA(D/H)-box RNA helicase homologs of human DDX3, DDX5 and DDX6, translation initiation factors, ribosomal proteins and transport factors. Our functional studies revealed that depletion of DDX3 helicases enhances the accumulation of NMD target reporterm RNAs but does not result in increased protein levels. In contrast, silencing of DDX6 group leads to decreased accumulation of the NMD substrate. The inhibitory effect of DDX6-like helicases on NMD was confirmed by transient over-expression of RH12 helicase. These results indicate that DDX3 and DDX6 helicases in plants have a direct and opposing contribution to NMD and act as functional NMD factors.

The study uses global quantitative proteomics to investigate the molecular mechanisms behind the induction of endothelial-mesenchymal transition (EndMT) by transforming growth factor–β (TGF-β). Orbitrap Velos mass spectrometers and iTRAQ – a labeling-based analysis were used to perform a global and quantitative comparison of two proteomes of Human Microvascular Endothelial Cells-1 (HMEC-1) treated or not treated by TGF-β1. iTRAQ analysis identified 43 differentially-expressed proteins in the early stages of EndMT induced by TGF-β1. From 5522 identified proteins, 26 were downregulated and 17 were upregulated, including proteins such as palladin, POTE I, torsin A and nucleoporin (NDC1). Further analysis of palladin revealed its increased mRNA and protein expression in response to TGF-β and Snail transcription factor. Our findings demonstrate that the newly- identified proteins may be involved in early stages of biological processes leading to EndMT. Biological Significance: Endothelial to mesenchymal transition is a possible source of myofibroblasts, which play a crucial role in the pathogenesis of fibrosis. EndMT participate in tissue fibrotic processes in various organs. TGF-β family growth factors are involved in the initiation of EndMT. The intracellular cascades activated by TGF-β that result in the remarkable phenotypic change of endothelial cells to mesenchymal cells have not been entirely elucidated. The downstream signaling pathway initiated by TGF-β resulted in a strong upregulation of the Snail1 transcriptional repressor. Our proteomics data demonstrated that TGF-β -induced EndMT leads to alterations in protein profiles, more specifically, the upregulation of palladin. This upregulation is mediated by Snail transcription factor and GSK-3 β signaling kinase. Our results also suggest that palladin could be considered a new biomarker in the early stages of cellular transdifferentiation, eventually leading to endothelial-mesenchymal transition.