Post-translational Regulation Research Articles

Proteome-wide forced interactions reveal a functional map of cell-cycle phospho-regulation in S. cerevisiae

ABSTRACT Dynamic protein phosphorylation and dephosphorylation play an essential role in cell cycle progression. Kinases and phosphatases are generally highly conserved across eukaryotes, underlining their importance for post-translational regulation of substrate proteins. In recent years, advances in phospho-proteomics have shed light on protein phosphorylation dynamics throughout the cell cycle, and ongoing progress in bioinformatics has significantly improved annotation of specific phosphorylation events to a given kinase. However, the functional impact of individual phosphorylation events on cell cycle progression is often unclear. To address this question, we used the Synthetic Physical Interactions (SPI) method, which enables the systematic recruitment of phospho-regulators to most yeast proteins. Using this method, we identified several putative novel targets involved in chromosome segregation and cytokinesis. The SPI method monitors cell growth and, therefore, serves as a tool to determine the impact of protein phosphorylation on cell cycle progression.

FUT8 Regulates Cerebellar Neurogenesis and Development Through Maintaining the Level of Neural Cell Adhesion Molecule Cntn2.

Core fucosylation at N-glycans, which is uniquely catalyzed by fucosyltransferase FUT8, plays essential roles in post-translational regulation of protein function. Aberrant core fucosylation leads to neurological disorders in individuals with congenital glycosylation disorders (CDG). However, the underlying mechanisms for these neurological defects remain largely unknown. In this study, we have showed that FUT8 and fucosylation are abundant in cerebellum. Specific deletion of Fut8 in cerebellar granule neuron progenitors (GNPs) results in the impaired proliferation and differentiation of GNPs, as well as the compromised neuronal development, synaptic physiology and motor coordination. Mechanistically, we have showed that Fut8 deficiency reduced Contactin 2 (Cntn2) expression, a member of neural cell adhesion molecules (NCAMs). Furthermore, ectopic Cntn2 can rescue the neuronal defects induced by Fut8 deficiency. Collectively, our study has revealed the important roles of FUT8 and core fucosylation in regulating cerebellar development and function through modulating Cntn2 expression.

In Vivo Quantification of Surfactin Nonribosomal Peptide Synthetase Complexes in Bacillus subtilis.

Surfactin, a potent biosurfactant produced by Bacillus subtilis, is synthesized using a non-ribosomal peptide synthetase (NRPS) encoded by the srfAA-AD operon. Despite its association with quorum sensing via the ComX pheromone, the dynamic behavior and in vivo quantification of the NRPS complex remain underexplored. This study established an in vivo quantification system using fluorescence labeling to monitor the availability of surfactin-forming NRPS subunits (SrfAA, SrfAB, SrfAC, and SrfAD) during bioprocesses. Four Bacillus subtilis sensor strains were constructed by fusing these subunits with the megfp gene, resulting in strains BMV25, BMV26, BMV27, and BMV28. These strains displayed growth and surfactin productivity similar to those of the parental strain, BMV9. Fluorescence signals indicated varying NRPS availability, with BMV27 showing the highest and BMV25 showing the lowest relative fluorescence units (RFUs). RFUs were converted to the relative number of NRPS molecules using open-source FPCountR package. During bioprocesses, NRPS availability peaked at the end of the exponential growth phase and declined in the stationary phase, suggesting reduced NRPS productivity under nutrient-limited conditions and potential post-translational regulation. This study provides a quantitative framework for monitoring NRPS dynamics in vivo, offering insights into optimizing surfactin production. The established sensor strains and quantification system enable the real-time monitoring of NRPS availability, aiding bioprocess optimization for industrial applications of surfactin and potentially other non-ribosomal peptides.

Phosphorylation of cytochrome c at tyrosine 48 finely regulates its binding to the histone chaperone SET/TAF-Iβ in the nucleus.

Post-translational modifications (PTMs) of proteins are ubiquitous processes present in all life kingdoms, involved in the regulation of protein stability, subcellular location and activity. In this context, cytochrome c (Cc) is an excellent case study to analyze the structural and functional changes induced by PTMS as Cc is a small, moonlighting protein playing different roles in different cell compartments at different cell-cycle stages. Cc is actually a key component of the mitochondrial electron transport chain (ETC) under homeostatic conditions but is translocated to the cytoplasm and even the nucleus under apoptotic conditions and/or DNA damage. Phosphorylation does specifically alter the Cc redox activity in the mitochondria and the Cc non-redox interaction with apoptosis-related targets in the cytoplasm. However, little is known on how phosphorylation alters the interaction of Cc with histone chaperones in the nucleus. Here, we report the effect of Cc Tyr48 phosphorylation by examining the protein interaction with SET/TAF-Iβ in the nuclear compartment using a combination of molecular dynamics simulations, biophysical and structural approaches such as isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) and in cell proximity ligation assays. From these experiments, we infer that Tyr48 phosphorylation allows a fine-tuning of the Cc-mediated inhibition of SET/TAF-Iβ histone chaperone activity in vitro. Our findings likewise reveal that phosphorylation impacts the nuclear, stress-responsive functions of Cc, and provide an experimental framework to explore novel aspects of Cc post-translational regulation in the nucleus.

DbPTM 2025 update: comprehensive integration of PTMs and proteomic data for advanced insights into cancer research.

Post-translational modifications (PTMs) are essential for modulating protein function and influencing stability, activity and signaling processes. The dbPTM 2025 update significantly expands the database to include over 2.79 million PTM sites, of which 2.243 million are experimentally validated from 48 databases and over 80 000 research articles. This version integrates proteomic data from 13 cancer types, with a particular focus on phosphoproteomic data and kinase activity profiles, allowing the exploration of personalized phosphorylation patterns in tumor samples. Integrating kinase-substrate phosphorylations with E3 ligase-substrate interactions, dbPTM 2025 provides a detailed map of PTM regulatory networks, offering insights into cancer-specific post-translational regulations. This update also includes advanced search capabilities, enabling users to efficiently query PTM data across species, PTM typesand modified residues. The platform's new features-interactive visualization tools and streamlined data downloads-allow researchers to access and analyze PTM data easily. dbPTM 2025 also enhances functional annotations, regulatory networks and disease associations, broadening its application for cancer research and the study of disease-associated PTMs. Through these enhancements, dbPTM 2025 is a comprehensive, user-friendly resource, facilitating the study of PTMs and their roles in cancer research. The database is now freely accessible at https://biomics.lab.nycu.edu.tw/dbPTM/.

Protein profile changes during priming explants to embryogenic response in Coffea canephora: identification of the RPN12 proteasome subunit involved in the protein degradation.

Plant somatic embryogenesis encompasses somatic cells switch into embryogenic cells that can later produce somatic embryos with the ability to produce plantlets. Previously, we defined in vitro culture settings for the somatic embryogenesis process of Coffea canephora that comprise adequate plantlets with auxin plus cytokinin followed by cut-leaf explant cultivation with cytokinin, producing embryos with the ability to regenerate plantlets. Here, we confirmed that cultivating cut-leaf explants with cytokinin is sufficient to promote somatic embryos proliferation and the high yield of somatic embryos in the protocol requires adequate plantlets with auxin plus cytokinin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels reveal auxin-plus cytokinin-dependent regulated proteins in plantlets with up and down abundance. Chitinase A class III, proteins involved in the metabolism and folding of proteins, photosynthesis, antioxidant activity, and chromatin organization were identified. The RPN12 protein, which is a subunit of the proteasome 26S, has an abundance that is not associated with transcript changes, suggesting post-translational regulation.

Building permits-control of type IV pilus assembly by PilB and its cofactors.

Many bacteria produce type IV pili (T4P), surfaced-exposed protein filaments that enable cells to interact with their environment and transition from planktonic to surface-adapted states. T4P are dynamic, undergoing rapid cycles of filament extension and retraction facilitated by a complex protein nanomachine powered by cytoplasmic motor ATPases. Dedicated assembly motors drive the extension of the pilus fiber into the extracellular space, but like any machine, this process is tightly organized. These motors are coordinated by various ligands and binding partners, which control or optimize their functional associations with T4P machinery before cells commit to the crucial first step of building a pilus. This review focuses on the molecular mechanisms that regulate T4P extension motor function. We discuss secondary messenger-dependent transcriptional or post-translational regulation acting both directly on the motor and through protein effectors. We also discuss the recent discoveries of naturally occurring extension inhibitors as well as alternative mechanisms of pilus assembly and motor-dependent signaling pathways. Given that T4P are important virulence factors for many bacterial pathogens, studying these motor regulatory systems will provide new insights into T4P-dependent physiology and efficient strategies to disable them.

PARylation of GCN5 by PARP1 mediates its recruitment to DSBs and facilitates both HR and NHEJ Repair

Efficient DNA double strand break (DSB) repair is necessary for genomic stability and determines efficacy of DNA damaging cancer therapeutics. Spatiotemporal dynamics and post-translational modifications of repair proteins at DSBs dictate repair efficacy. Here, we identified a non-canonical function of GCN5 in regulating both HR and NHEJ repair post genotoxic stress. Mechanistically, genotoxic stress induced GCN5 recruitment to DSBs. GCN5 PARylation by PARP1 was essential for its recruitment, acetyltransferase activity and DSB repair function. Liquid chromatography-mass spectrometry (LC–MS) identified DNA-PKcs as part of GCN5 interactome. In-vitro acetyltransferase assays revealed that GCN5 acetylates DNA-PKcs at K3241 residue, a prerequisite for DNA-PKcs S2056 phosphorylation and DSB recruitment. Alongside, ChIP-qPCR revealed GCN5 mediates transcription of PRKDC via H3K27Ac acetylation in its promoter region (− 710 to − 554). Genetic perturbation of GCN5 also decreased CHEK1, NBN1, TP53BP1, POL-L transcription and abrogated ATM, BRCA1 activation. Accordingly, GCN5 loss led to persistent ɣ-H2AX foci formation, compromised in-vivo HR-NHEJ and caused GBM radio-sensitization. Importantly, PARP1 inhibition phenocopied GCN5 loss. Together, this study identifies an untraversed DSB repair function of GCN5 and provides mechanistic insights into transcriptional as well as post-translational regulation of pivotal HR-NHEJ factors. Alongside, it highlights the translational importance of PARP1-GCN5 axis in mediating GBM radio-resistance.

Circulating microRNAs associated with bronchodilator response in childhood asthma

BackgroundBronchodilator response (BDR) is a measure of improvement in airway smooth muscle tone, inhibition of liquid accumulation and mucus section into the lumen in response to short-acting beta-2 agonists that varies among asthmatic patients. MicroRNAs (miRNAs) are well-known post-translational regulators. Identifying miRNAs associated with BDR could lead to a better understanding of the underlying complex pathophysiology.ObjectiveThe purpose of this study is to identify circulating miRNAs associated with bronchodilator response in asthma and decipher possible mechanism of bronchodilator response variation.MethodsWe used available small RNA sequencing on blood serum from 1,134 asthmatic children aged 6 to 14 years who participated in the Genetics of Asthma in Costa Rica Study (GACRS). We filtered the participants into the highest and lowest bronchodilator response (BDR) quartiles and used DeSeq2 to identify miRNAs with differential expression (DE) in high (N = 277) vs. low (N = 278) BDR group. Replication was carried out in the Leukotriene modifier Or Corticosteroids or Corticosteroid-Salmeterol trial (LOCCS), an adult asthma cohort. The putative target genes of DE miRNAs were identified, and pathway enrichment analysis was performed.ResultsWe identified 10 down-regulated miRNAs having odds ratios (OR) between 0.37 and 0.76 for a doubling of miRNA counts and one up-regulated miRNA (OR = 2.26) between high and low BDR group. These were assessed for replication in the LOCCS cohort, where two miRNAs (miR-200b-3p and miR-1246) were associated. Further, functional annotation of 11 DE miRNAs were performed as well as of two replicated miRs. Target genes of these miRs were enriched in regulation of cholesterol biosynthesis by SREBPs, ESR-mediated signaling, G1/S transition, RHO GTPase cycle, and signaling by TGFB family pathways.ConclusionMiRNAs miR-1246 and miR-200b-3p are associated with both childhood and adult asthma BDR. Our findings add to the growing body of evidence that miRNAs play a significant role in the difference of asthma treatment response among patients as it points to genomic regulatory machinery underlying difference in bronchodilator response among patients.Trial registrationLOCCS cohort [ClinicalTrials.gov number NCT00156819, Registration date 20050912], GACRS cohort [ClinicalTrials.gov number NCT00021840].

Identification of the fructose 1,6-bisphosphate aldolase (FBA) family genes in maize and analysis of the phosphorylation regulation of ZmFBA8

Fructose 1,6-bisphosphate aldolase (FBA) is a class of aldolase that functions as enzyme participating in carbohydrate metabolism of the Calvin-Benson cycle, gluconeogenesis, and glycolysis, and also as non-enzymatic protein involving in protein binding, gene transcription, signal transduction. FBAs have been identified in a few plant species, however, limited information is known regarding FBA family genes, their biological functions and posttranslational regulations in maize (Zea mays). In this study, nine class I FBAs (ZmFBA1 to ZmFBA9) and one class II FBA (ZmFBA10) in maize were identified. Phosphoproteomic analysis further revealed that multiple ZmFBAs were phosphorylated. We showed that phosphorylation at Ser32 in ZmFBA8 inhibited its FBP binding and enzyme activity. Loss of ZmFBA8 function reduced the growth of maize seedlings. Our results suggest that the phosphorylation is an important regulatory mechanism of ZmFBA8 function.

HMGA2 regulates GPX4 expression and ferroptosis in prostate cancer cells

Prostate cancer (PCa) remains a significant global health burden and an increase in oxidative stress is associated with cancer progression. High Mobility Group A2 (HMGA2), a chromatin architectural protein, increases oxidative stress and promotes sensitivity to ferroptosis inducers, however, the mechanism is unknown. We investigated the role of HMGA2 in GPX4 regulation and the impact on cellular responses to oxidative stress and ferroptosis sensitivity. We conducted UALCAN database analysis, western blot analysis, and lipid peroxidation assays to determine the relationship between HMGA2 and GPX4 and the levels of lipid reactive oxygen species in a panel of PCa cell lines, including an enzalutamide-resistant cancer cell line (C4–2B MDVR). Our results show an inverse relationship between HMGA2 and GPX4 expression with high HMGA2 and low GPX4 expression associated with higher Gleason score and lower survival probability in prostate adenocarcinoma (PRAD) patients, while low/moderate HMGA2 expression is positively associated with increased GPX4 expression and higher survival probability. Cell lines showed a moderately negative but not statistically significant correlation between HMGA2 and GPX4 expression, however, PC3 and DU145 PCa cells display higher lipid peroxides concomitant with higher endogenous levels of HMGA2 and low GPX4. Overexpression of wild-type HMGA2 in LNCaP and 22Rv1 cells leads to higher HMGA2 expression compared to Neo control and is associated with higher SLC7A11 and GPX4 expression, while interestingly truncated HMGA2 overexpression in LNCaP and 22Rv1 cells coincides with higher HMGA2 and reduced GPX4 expression, leading to increased lipid peroxides and susceptibility to ferroptosis. Overexpression of wild-type and truncated HMGA2 in 22Rv1 cells increases SLC7A11 mRNA yet differing GPX4 protein expression suggests posttranslational regulation of GPX4. Moreover, enzalutamide-resistant C4–2B MDVR cells display higher HMGA2 levels compared to C4–2B cells, as well as sensitivity to RSL3 ferroptosis inducer, which is partially reversed by ferroptosis inhibitor, ferrostatin-1. Interestingly, GPX4 expression is higher in C4–2B MDVR cells compared to C4–2B, and HMGA2 knockdown further increases its expression but does not significantly alter its susceptibility to ferroptosis. In conclusion, our study shows that HMGA2 regulation of GPX4 expression is complex and truncated HMGA2 downregulates GPX4 and increases lipid peroxides. Moreover, HMGA2-expressing cells including enzalutamide-resistant cells are susceptible to RSL-3-induced ferroptosis. Thus, ferroptosis sensitivity offers promising insights for the development of targeted therapeutic interventions for aggressive PCa.

Understanding fungal and plant active urea transport systems: Keys from Aspergillus nidulans and beyond

Urea is present in all ecosystems, as a result of the metabolism of different organisms and also of human activity, being the world's most common form of nitrogen fertilizer. Fungi and plants can use urea as a nitrogen source, taking it up from the environment through specialized active transport proteins. These proteins belong to a subfamily of urea/H+ symporters included in the Solute:Sodium Symporter (SSS) family of transporters. In this review we summarize the current knowledge on this group of transporters, based on our previous studies on Aspergillus nidulans UreA. We delve into its transcriptional and post-translational regulation, structure-function relationships, transport mechanism, and certain aspects of its biogenesis. Recent findings suggest that this urea transporter subfamily is more expanded than originally thought, with representatives found in organisms as diverse as Archaea and mollusks, which raises questions on evolutionary aspects. A. nidulans ureA knockout strains provide a valuable platform for expressing urea transporters from diverse sources, facilitating their characterization and functional analysis. In this context, given the close relationship between plant and fungal active urea transporters, this knowledge could serve to develop strategies to improve the efficiency of applied urea as fertilizer.

TRAF7 determines circadian period through ubiquitination and degradation of DBP.

D-site binding protein, DBP, is a clock-controlled transcription factor and drives daily rhythms of physiological processes through the regulation of an array of genes harboring a DNA binding motif, D-box. DBP protein levels show a circadian oscillation with an extremely robust peak/trough ratio, but it is elusive how the temporal pattern is regulated by post-translational regulation. In this study, we show that DBP protein levels are down-regulated by the ubiquitin-proteasome pathway. Analysis using 19 dominant-negative forms of E2 enzymes have revealed that UBE2G1 and UBE2T mediate the degradation of DBP. A proteomic analysis of DBP-interacting proteins and database screening have identified Tumor necrosis factor Receptor-Associated Factor 7 (TRAF7), a RING-type E3 ligase, that forms a complex with UBE2G1 and/or UBE2T. Ubiquitination analysis have revealed that TRAF7 enhances K48-linked polyubiquitination of DBP in cultured cells. Overexpression of TRAF7 down-regulates DBP protein level, while knockdown of TRAF7 up-regulates DBP in cultured cells. Knockout of TRAF7 in NIH3T3 cells have revealed that TRAF7 mediates the time-of-the-day-dependent regulation of DBP levels. Furthermore, TRAF7 has a period-shortening effect on the cellular clock. Together, TRAF7 plays an important role in circadian clock oscillation through destabilization of DBP.

Translation Initiation Factor-2S2 (eIF2S2) Contributes to Cervical Carcinogenesis by Inhibiting the TGF-β/SMAD4 Signaling Pathway.

The deregulation of protein translational machinery and the oncogenic role of several translation initiation factors have been extensively investigated. This study aimed to investigate the role of eukaryotic translation initiation factor 2S2 (eIF2S2, also known as eIF2β) in cervical carcinogenesis. Immunohistochemical analysis of human cervical carcinoma tissues revealed a stage-specific increase in eIF2S2 expression. The knockdown of eIF2S2 in human cervical cancer (SiHa) cells significantly reduced growth and migration properties, whereas its overexpression demonstrated the opposite effect. Immunoprecipitation and Bimolecular fluorescence complementation (BiFC) assay confirmed the previous photo array finding of the interaction between eIF2S2 and SMAD4 to understand the tumorigenic mechanism of eIF2S2. The results indicated that the N-terminus of eIF2S2 interacts with the MH-1 domain of SMAD4. The interaction effect between eIF2S2 and SMAD4 was further evaluated. The knockdown of eIF2S2 increased SMAD4 expression in cervical cancer cells without changing SMAD4 mRNA expression, whereas transient eIF2S2 overexpression reduced SMAD4 expression. This indicates the possibility of post-translational regulation of SMAD4 expression by eIF2S2. Additionally, eIF2S2 overexpression was confirmed to weaken the expression and/or promoter activity of p15 and p27, which are SMAD4-regulated antiproliferative proteins, by reducing SMAD4 levels. Therefore, our study indicated the pro-tumorigenic role of eIF2S2, which diminishes both SMAD4 expression and function as a transcriptional factor in cervical carcinogenesis.

Dysregulation of RAS proteostasis by autosomal-dominant LZTR1 mutation induces Noonan syndrome-like phenotypes in mice.

Leucine-zipper-like post translational regulator 1 (LZTR1) is a member of the BTB-Kelch superfamily, which regulates the RAS proteostasis. Autosomal dominant (AD) mutations in LZTR1 have been identified in patients with Noonan syndrome (NS), a congenital anomaly syndrome. However, it remains unclear whether LZTR1 AD mutations regulate the proteostasis of the RAS subfamily molecules or cause NS-like phenotypes in vivo. To elucidate the pathogenesis of LZTR1 mutations, we generated two novel LZTR1 mutation knock-in mice (Lztr1G245R/+ and Lztr1R409C/+), which correspond to the human p.G248R and p.R412C mutations, respectively. LZTR1-mutant male mice exhibit low birth weight, distinctive facial features, and cardiac hypertrophy. Cardiomyocyte size and the expression of RAS subfamily members, including MRAS and RIT1, were significantly increased in the left ventricles (LVs) of mutant male mice. LZTR1 AD mutants did not interact with RIT1 and functioned as dominant-negative forms of wild-type LZTR1. Multi-omics analysis revealed that the MAPK signaling pathway was activated in the LVs of mutant mice. Treatment with the MEK inhibitor trametinib ameliorated cardiac hypertrophy in mutant male mice. These results suggest that MEK/ERK pathway is a therapeutic target for NS-like phenotype resulting from dysfunction of RAS proteostasis by LZTR1 AD mutations.

The BpPP2C-BpMADS11-BpERF61 signaling confers drought tolerance in Betula platyphylla.

Plant MADS-box proteins are vital for abiotic stress tolerance, yet their mechanisms for responding to drought remain poorly understood. Here, we investigated the drought tolerance mechanism of a MADS-box protein (BpMADS11) from birch (Betula platyphylla) using immunoprecipitation, Western blotting, yeast two-hybrid, yeast one-hybrid, ChIP, RNA-seq, and dual-luciferase assays to explore post-translational modifications, protein interactions, and gene regulation. Birch plants overexpressing BpMADS11 exhibited enhanced drought tolerance, while knockout lines displayed reduced tolerance. Under drought conditions, BpMADS11 interacts with protein phosphatase 2C22 (BpPP2C22), which dephosphorylates BpMADS11. Birch plants that overexpress BpMADS11 and lack BpPP2C22 show significantly reduced drought tolerance compared with those that only overexpress BpMADS11. BpMADS11 regulates the expression of BpERF61 by binding to CArG-box in its promoter. The dephosphorylated BpMADS11 exhibits increased DNA binding ability and increased expression of BpERF61. Like BpMADS11, birch plants overexpressing BpERF61 show improved drought tolerance, while those with BpERF61 knockout exhibit decreased tolerance. BpERF61 binds to specific DNA motifs including 'CACGTG' (G-box), 'GGGCCCC', and 'TTGGAT' to regulate the genes related to drought stress. Collectively, BpMADS11 undergoes dephosphorylation through its interaction with BpPP2C22, prompting the expression of BpERF61. Subsequently, BpERF61 regulates downstream genes by binding to specific DNA motifs, thereby enhancing drought tolerance.

Characterizing Leishmania infantum-induced resistance to trivalent stibogluconate (SbIII) through deep proteomics

Leishmania infantum belongs to the L. donovani complex, which includes species associated with visceral leishmaniasis. Traditionally, antimonial compounds have served as the primary antiparasitic treatment for all clinical forms of leishmaniasis. However, the global spread of resistance to these compounds has posed a significant challenge in the treatment in some regions. In this study, we aimed to investigate resistance to trivalent sodium stibogluconate in vitro using promastigotes from a wild strain of L. infantum. We compared the growth rates and proteomic profiles of wild-type and resistant line conducting label-free quantitative mass spectrometry-based proteomic analyses. Statistical and bioinformatics analyses were employed to evaluate the significance of protein concentration changes, protein identity annotation, GO term analysis, biosynthetic pathways, and protein-protein interactions. Our findings revealed that the resistant line displayed a notable reduction in growth rate. Proteomic data unveiled similar protein concentrations per cell in both groups but with differing molecule copy numbers. We identified 165 proteins with increased concentration, these were associated with transcription and translation activities, lipid metabolism, energy metabolism, and peroxisome biogenesis. In the decreased protein groups were 56 proteins linked to metal acquisition and metabolism, particularly iron. These results suggest a novel perspective on antimonial resistance, highlighting the importance of post-transcriptional and post-translational regulation, alongside energy expenditure compensation and alterations in organelle membrane lipid composition in antimonial-resistant parasites. Overall, our study provides insights into the proteomic profile of stibogluconate-resistant strain, contributing to our general understanding of the complex landscape of antiparasitic resistance in L. infantum. SignificanceSpecies within the Leishmania donovani complex are implicated in cases of visceral leishmaniasis in the world. Leishmania infantum is a species that predominates in regions spanning the Mediterranean Basin, the Middle East, Central Asia, South and Central America. Antimonials were the first treatment for leishmaniasis, however in the last decades, the resistance has emerged in subregions like India, where it is not a therapeutic option. In contrast, sodium stibogluconate (SbIII) remains the first-line treatment in the Americas. Unfortunately, the emergence of resistance has outpaced the development of new therapeutic options, thereby becoming a critical point in the struggle against the disease. In this study we performed an in-depth proteomic analysis with liquid chromatography mass-mass spectrometry (LC-MS/MS) on L. infantum with Sb-induced resistance in vitro. Results showed a complex proteomic adaptation in the resistant line, involving transcriptional and translational proteins, energy compensation, and homeostasis maintenance. These insights contribute to understanding the molecular adaptation in the parasite and provide information to new investigations related to therapeutics development.

A hierarchical ubiquitination-mediated regulatory module controls bamboo lignin biosynthesis

Abstract The lignocellulosic feedstock of woody bamboo shows promising potential as an alternative to conventional wood, attributed to its excellent properties. The content and distribution of lignin serve as the foundation of these properties. While the regulation of lignin biosynthesis in bamboo has been extensively studied at the transcriptional level, its posttranslational control has remained poorly understood. This study provides a ubiquitinome dataset for moso bamboo (Phyllostachys edulis), identifying 13015 ubiquitinated sites in 4849 unique proteins. We further identified Kelch repeat F-boxprotein 9 (PeKFB9) that plays a negative role in lignin biosynthesis. Heterologous expression of PeKFB9 resulted in reduced accumulation of lignin and decreased phenylalanine ammonia-lyase (PAL) activities. Both in vitro and in vivo assays identified interaction between PeKFB9 and PePAL10. Further examination revealed that SCFPeKFB9 mediated the ubiquitination and degradation of PePAL10 via the 26S proteasome pathway. Moreover, PebZIP28667 could bind to the PePAL10 promoter to significantly inhibit its transcription, and ubiquitination of PebZIP28667 weakened this inhibition. Collectively, our findings reveal a PeKFB9-PePAL10/PebZIP28667-PePAL10 module that acts as a negative regulator of lignin biosynthesis. This study advances our understanding of posttranslational regulation in plant lignification, which will facilitate the improvement of the properties of bamboo wood and the breeding of varieties.

PIN1 is a novel interaction partner and a negative upstream regulator of the transcription factor NFIB.

NFIB is a transcription factor of the Nuclear Factor One (NFI) family that is essential for embryonic development. Post-translational control of NFIB or its upstream regulators have not been well characterized. Here, we show that PIN1 binds NFIB in a phosphorylation-dependent manner, via its WW domain. PIN1 interacts with the well-conserved N-terminal domains of all NFIs. Moreover, PIN1 attenuates the transcriptional activity of NFIB; this attenuation requires substrate binding by PIN1 but not its isomerase activity. Paradoxically, we found stabilization of NFIB by PIN1. We propose that PIN1 represses NFIB function not by regulating its abundance but by inducing a conformational change. These results identify NFIB as a novel PIN1 target and posit a role for PIN1 in post-translational regulation of NFIB and other NFIs.

The proteomic landscape and temporal dynamics of mammalian gastruloid development.

Gastrulation is the highly coordinated process by which the early embryo breaks symmetry, establishes germ layers and a body plan, and sets the stage for organogenesis. As early mammalian development is challenging to study in vivo, stem cell-derived models have emerged as powerful surrogates, e.g. human and mouse gastruloids. However, although single cell RNA-seq (scRNA-seq) and high-resolution imaging have been extensively applied to characterize such in vitro embryo models, a paucity of measurements of protein dynamics and regulation leaves a major gap in our understanding. Here, we sought to address this by applying quantitative proteomics to human and mouse gastruloids at four key stages of their differentiation (naïve ESCs, primed ESCs, early gastruloids, late gastruloids). To the resulting data, we perform network analysis to map the dynamics of expression of macromolecular protein complexes and biochemical pathways, including identifying cooperative proteins that associate with them. With matched RNA-seq and phosphosite data from these same stages, we investigate pathway-, stage- and species-specific aspects of translational and post-translational regulation, e.g. finding peri-gastrulation stages of human and mice to be discordant with respect to the mitochondrial transcriptome vs. proteome, and nominating novel kinase-substrate relationships based on phosphosite dynamics. Finally, we leverage correlated dynamics to identify conserved protein networks centered around congenital disease genes. Altogether, our data (https://gastruloid.brotmanbaty.org/) and analyses showcase the potential of intersecting in vitro embryo models and proteomics to advance our understanding of early mammalian development in ways not possible through transcriptomics alone.