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Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

Chronic Kidney Disease (CKD) is associated with heightened risk of thrombosis. Prescription of anticoagulants is key to manage it; however, CKD patients have shown an increased risk of bleeding under anticoagulation therapy compared to non-CKD patients. We hypothesized that the sex could modify the metabolism of indoxyl sulfate (IS), a uremic toxin and Apixaban. Our intoxication model shows that higher doses of IS and apixaban accumulate in the plasma of female mice because of expression differences in efflux transporters and cytochromes in the liver, ileum and kidneys, when compared to males. Furthermore, we found that accumulation of apixaban in females contributes to increased bleeding. Transcriptional analysis of liver samples revealed elevated Sult1a1 but reduced Abcg2 and Cyp3a11 in female mice, while in the kidneys the expression rates of Oat1 and Oat3 were respectively lower and higher than those observed in males, potentially affecting drug clearance. Whole proteomics liver analysis confirmed the previous transcriptional results at the protein level and revealed that sex had a major influence in regulating both coagulation and drug metabolism pathways. Thus, our findings underline the need for inclusive clinical and preclinical trials to accurately reflect sex-specific metabolic variations, and to consider CKD-specific changes to optimize dosing, minimize side effects, and improve patient outcomes.

Splicing Factor 3b Subunit 1 (SF3B1), a core component of the spliceosome, undergoes dynamic phosphorylation and dephosphorylation during the splicing cycle to regulate pre-mRNA splicing. Twenty-eight threonine/proline repeats are phosphorylated by CDK11 during spliceosome activation and remain phosphorylated in the catalytically active spliceosomes. The function of phosphorylated SF3B1 (P-SF3B1), and the identity of spliceosomes stalled by CDK11 inhibition remain unclear. Using quantitative proteomics of chromatin-associated spliceosomes, we identify a previously uncharacterized intermediate complex B OTS964 , arrested by CDK11 inhibitor OTS964, that incorporates the nineteen-related (NTR) but not nineteen (NTC) complex. iCLIP-seq revealed that P-SF3B1 engages with the U6 snRNA internal stem-loop (ISL), suggesting a potential role in stabilizing the RNA catalytic core. We further demonstrate that P-SF3B1 is recognized by forkhead-associated (FHA) domain of SNIP1, which promotes recruitment of retention and splicing (RES) complex during spliceosome activation. Acute SNIP1 depletion disrupts RES incorporation, causes widespread splicing defects, and promotes hyperphosphorylation of SF3B1 by CDK11. Mutations in SNIP1 FHA domain, including the neurodevelopmental disorder-associated E366G variant, impair P-SF3B1 binding, pre-mRNA splicing, and cell viability. Together, these findings uncover the phosphorylation-dependent CDK11/P-SF3B1/SNIP1 signaling axis that is critical for pre-mRNA splicing and cellular proliferation and provide a mechanistic insight into its dysregulation in disease. Cells rely on tightly controlled RNA splicing to ensure proper gene expression. Here, the authors show that CDK11-driven phosphorylation of SF3B1 recruits SNIP1 and the RES complex to activate the spliceosome, uncovering a signaling pathway essential for accurate splicing, cell survival, and disease prevention.

Background As in animals, cell–cell communication plays a pivotal role in male–female recognition during plant sexual reproduction. Prelaid peptides secreted from the female reproductive tissues guide pollen tubes towards ovules for fertilization. However, the elaborate mechanisms for this dialogue have remained elusive, particularly from the male perspective. Results We performed genome-wide quantitative liquid chromatography–tandem mass spectrometry analysis of a pistil-stimulated pollen tube secretome and identified 801 pollen tube-secreted proteins. Interestingly, in silico analysis reveals that the pollen tube secretome is dominated by proteins that are secreted unconventionally, representing 57 % of the total secretome. In support, we show that an unconventionally secreted protein, translationally controlled tumor protein, is secreted to the apoplast. Remarkably, we discovered that this protein could be secreted by infiltrating through the initial phases of the conventional secretory pathway and could reach the apoplast via exosomes, as demonstrated by co-localization with Oleisin1 exosome marker. We demonstrate that translationally controlled tumor protein-knockdown Arabidopsis thaliana plants produce pollen tubes that navigate poorly to the target ovule and that the mutant allele is poorly transmitted through the male. Further, we show that regulators of the endoplasmic reticulum–trans-Golgi network protein secretory pathway control secretion of Nicotiana tabacum Pollen tube-secreted cysteine-rich protein 2 and Lorelei-like GPI-anchor protein 3 and that a regulator of endoplasmic reticulum–trans-Golgi protein translocation is essential for pollen tube growth, pollen tube guidance and ovule-targeting competence. Conclusions This work, the first study on the pollen tube secretome, identifies novel genome-wide pollen tube-secreted proteins with potential functions in pollen tube guidance towards ovules for sexual reproduction. Functional analysis highlights a potential mechanism for unconventional secretion of pollen tube proteins and reveals likely regulators of conventional pollen tube protein secretion. The association of pollen tube-secreted proteins with marker proteins shown to be secreted via exosomes in other species suggests exosome secretion is a possible mechanism for cell–cell communication between the pollen tube and female reproductive cells.

RNF43 is an E3 ubiquitin ligase and known negative regulator of WNT/β-catenin signaling. We demonstrate that RNF43 is also a regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1, and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation. These activities of RNF43 are physiologically relevant and block pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E and MEK inhibitors. Next, RNF43 acted as melanoma suppressor and improved response to targeted therapies in vivo. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have a worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.

Background Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson’s disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson’s disease patients. Methods Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis ’ early development. Results Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo , in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Conclusions Our study shows for the first time that LRRK2 activates the WNT/PCP signaling pathway through its interaction to multiple WNT/PCP components. We suggest that LRRK2 regulates the balance between WNT/β-catenin and WNT/PCP signaling, depending on the binding partners. Since this balance is crucial for homeostasis of midbrain dopaminergic neurons, we hypothesize that its alteration may contribute to the pathophysiology of Parkinson’s disease.

The eIF4F translation initiation complex controls ERK MAPK signaling in malignant melanomas with BRAF and NRAS mutations. It also contributes to the development of melanoma resistance to therapies targeting BRAF and MEK kinases. Here, we uncovered a critical role for eIF4F in regulating the main cellular metabolic sensor, AMP-activated protein kinase (AMPK). In melanoma cells harboring the most common BRAF V600E mutation, ERK and AMPK pathway activities were reported as mutually exclusive. This is because BRAF-driven ERK activity negatively affects LKB1-mediated canonical AMPK activation. However, we observed that eIF4F inhibition can stimulate AMPK activity in melanoma cells, both in vitro and in vivo, despite concomitant ERK hyperactivation. Notably, the protein levels of LKB1 and its co-factor MO25 were sensitive to eIF4F inhibition, indicating a non-canonical LKB1-independent mechanism of AMPK activation. In a proteomic screen, we aimed to identify eIF4F roles in melanoma cell physiology beyond the MAPK pathway. We found that the eIF4F function is essential for maintaining cellular levels of key cell cycle and metabolic regulators, including CDK1, CDK2, TYMS, and UHRF1. Importantly, we also identified the protein phosphatase PP2A as a new eIF4F pathway target. Our subsequent analyses showed that inhibition or siRNA-mediated knockdown of PP2A increases AMPK activity in melanoma cells, independent of LKB1. This data shows that PP2A plays a significant role in regulating AMPK activity in melanoma. Thus, eIF4F inhibition not only impairs canonical AMPK activators but also downregulates PP2A, which negatively regulates AMPK dynamics. Collectively, our data highlight a dual role for eIF4F in controlling AMPK in BRAF -mutant melanoma cells. It maintains the canonical AMPK signaling pathway while simultaneously limiting the extent of AMPK activation via the eIF4F-PP2A-AMPK axis. Pharmacological inhibition of this axis can overcome the negative control of AMPK signaling by the ERK pathway. This suggests new therapeutic opportunities to disrupt melanoma growth.

Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (Nicotiana tabacum), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes. We demonstrated that the composition of the aforementioned complexes is not rigid and that numerous transcripts were redistributed among these complexes during pollen development, which may represent an important mechanism of translational regulation. Therefore, we defined the pollen sequestrome as a distinct and highly dynamic compartment for the storage of stable, translationally repressed transcripts and demonstrated its dynamics. We propose that EDTA/puromycin-resistant particle complexes represent aggregated nontranslating monosomes as the primary mediators of messenger RNA sequestration. Such organization is extremely useful in fast tip-growing pollen tubes, where rapid and orchestrated protein synthesis must take place in specific regions.

Some viroids—single-stranded, non-coding, circular RNA parasites of plants—are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (−) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming “comets” on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.