Biology subjects

Nawrocki, A.

Publications and source records attributed to Nawrocki, A..

11 recordsLinked to original sources

bioRxiv2026

STEP-PTMs: Sequential TMT-based Enrichment and Profiling of Post-Translational Modifications

Comprehensive characterization of protein abundance and multiple post-translational modifications (PTMs) from the same biological samples is essential for understanding cellular regulation and PTM crosstalk but remains analytically challenging. Here, we present STEP-PTM (Sequential Tag-based Enrichment of Post-Translational Modifications), a modular TMT-multiplexed workflow that enables integrated quantitative analysis of the proteome, metabolome and multiple PTM classes from a single peptide preparation. Proteins are digested, isobarically labeled using tandem mass tags (TMT), and combined into a single multiplexed peptide pool prior to sequential PTM enrichment, thereby minimizing technical variability, reducing sample requirements and facilitating direct quantitative integration across datasets. STEP-PTM supports flexible sequential enrichment of phosphopeptides, peptides containing free and reversibly modified cysteines, sialylated N-linked glycopeptides, lysine-acetylated peptides and S-palmitoylated peptides, while preserving non-modified peptides for global proteome analysis. PTM-specific database searches further improve identification confidence and quantitative accuracy, and the modular workflow can readily be adapted by incorporating or omitting enrichment modules according to the biological question. Application of STEP-PTM to TMT16-plex cerebral brain organoids enabled the quantification of 10,413 proteins, 2,969 metabolites, 19,655 phosphopeptides, 28,876 peptides containing reversibly modified cysteines, 9,723 peptides containing free cysteines, 1,716 intact sialylated N-linked glycopeptides and 771 lysine-acetylated peptides from the same biological samples. We further demonstrate the applicability of the workflow to multiple mouse tissues, highlighting its broad utility for integrated systems-level characterization of protein expression and PTM regulation across diverse biological models.

biochemistry↗

bioRxiv2026

GPMAW Glyco-Search: An Integrated Workflow for Identification and Validation of Intact Sialylated N-Glycopeptides

Comprehensive analysis of intact sialylated N-glycopeptides remains challenging because of their low abundance, extensive structural heterogeneity, and limited peptide backbone fragmentation during tandem mass spectrometry. Here, we present an integrated workflow for high-confidence identification of intact sialylated N-glycopeptides that combines selective TiO2 enrichment, dual LC-MS/MS analysis of intact and deglycosylated glycopeptides, and the GPMAW glyco-search platform based on high-accuracy mass mapping. Unlike most conventional glycoproteomics search engines, GPMAW uses experimentally identified deglycopeptides to constrain glycan assignment before matching intact glycopeptide precursor masses to candidate glycan compositions. Identifications were validated using diagnostic oxonium ions, glycopeptide-associated Y-ion fragments, and an experimentally derived glycopeptide score. In addition, GPMAW integrates an interactive spectrum annotation interface that enables rapid manual validation of candidate identifications through visualization of annotated Y-ion series, oxonium ions, and peptide fragments, allowing individual assignments to be readily accepted or rejected. The workflow was optimized using bovine fetuin, validated on standard glycoproteins, and applied to depleted human plasma, where more than 2800 unique intact sialylated N-glycopeptides were identified across hundreds of glycosites and glycoproteins. Moreover, more than 1000 unique N-glycopeptides were identified from only 1 L of plasma. Comparative analysis demonstrated that GPMAW glyco-search identified more confidently assigned intact sialylated N-glycopeptides than three widely used N-glycoproteomics search engines while maintaining high reproducibility and low false-positive rates following manual validation. Together, this workflow provides a robust, flexible, and accessible platform for large-scale, high-confidence characterization of intact N-glycopeptides and establishes experimentally constrained glycan composition assignment combined with interactive spectrum validation as an effective strategy for reducing ambiguity in N-glycoproteomics. HighlightsO_LIThe program "GPMAW glyco-search" enables high-accuracy mass mapping for confident identification of intact N-glycopeptides. C_LIO_LIIntegrated workflow combining TiO2 enrichment, dual LC-MS/MS of intact and deglycosylated glycopeptides and GPMAW glyco-search for intact sialylated N-glycopeptides. C_LIO_LIOptimized TiO2 enrichment provides >95% selective enrichment of sialylated N-glycopeptides from complex biological samples. C_LIO_LIInteractive spectrum annotation and Y-ion-based scoring enable rapid manual validation and high-confidence glycopeptide identification. C_LIO_LIGPMAW glyco-search confidently identified more intact sialylated N-linked glycopeptides compared to three established glycoproteomics search engines. C_LI

biochemistry↗

bioRxiv2026

Protein-state dysregulation and sex-specific neurodevelopmental signatures in schizophrenia forebrain organoids

Schizophrenia is highly heritable, yet the molecular mechanisms linking genetic risk to abnormal human brain development remain poorly understood. To address this, we generated dorsal forebrain organoids from 17 individuals with idiopathic schizophrenia and 17 age- and sex-matched controls and profiled them across multiple molecular layers, including single-nucleus transcriptomics, quantitative proteomics, metabolomics and deep post-translational modification (PTM) analysis. The organoids reproducibly modelled early cortical development and showed largely similar cellular composition between schizophrenia and control groups. Surprisingly, transcriptomic differences were relatively limited, with the strongest cell-type-specific changes observed in Cajal-Retzius neurons. In contrast, proteomic and particularly PTM-level analyses revealed widespread molecular disruption affecting pathways involved in neuronal migration, neurite development, synaptic function, protein kinase signalling, extracellular matrix organisation and lipid metabolism. Many of the earliest disease-associated changes emerged at the level of protein phosphorylation, consistent with altered neuronal maturation and neurite dynamics. At later developmental stages, schizophrenia organoids showed reduced abundance of synaptic proteins, fewer synaptic puncta and evidence of dysregulated retinoic acid and YAP1 signalling. Notably, most disease-associated alterations occurred independently of changes in transcript or protein abundance, indicating that key aspects of schizophrenia biology are encoded in protein state rather than expression level. These findings identify sex-specific dysregulation of protein state as a major molecular feature of schizophrenia and demonstrate the value of multi-layer proteomic approaches for uncovering disease mechanisms missed by transcriptomics alone.

neuroscience↗

bioRxiv2026

Dysregulation of miRNAs Drives Premature GABAergic Maturation and Early Neurodevelopmental Defects in Schizophrenia

BackgroundSchizophrenia (SCZ) is a severe neurodevelopmental disorder with numerous genetic risk loci. However, little is known about the molecular alterations that occur during brain development in SCZ, particularly regarding the role of microRNA (miRNA) mediated regulatory mechanisms. This gap in knowledge is largely due to the limited availability of developing human brain tissue. Patient-derived brain organoids offer a promising alternative model. Here we use 3D dorsal forebrain organoids (DFOs) to investigate miRNA dysregulation in SCZ. MethodsDFOs were generated from human induced pluripotent stem cells (hiPSCs) derived from six SCZ patients and five matched controls and cultured for 120 days. Multi-omics analyses, immunohistochemistry, and in situ hybridization were employed to characterize molecular and spatial features. ResultsDFOs recapitulated key molecular hallmarks of human cortical development. Nineteen miRNAs were differentially expressed in SCZ: nine associated with neural progenitor proliferation were downregulated and ten linked to neuronal differentiation and synaptic maturation were upregulated, reflecting a compressed developmental timeline. Among 77 dysregulated mRNAs, 55 were predicted miRNA targets. SCZ DFOs exhibited significant upregulation of GABAergic pathway genes accompanied by altered expression of their regulatory miRNAs, indicating premature GABAergic lineage specification. The disrupted miRNA-mRNA network converged on glutamatergic and dopaminergic development, synaptic organization, and extracellular matrix remodeling. ConclusionDysregulated miRNAs in SCZ DFOs disrupt neuronal differentiation, excitatory-inhibitory balance, and early circuit formation, implicating miRNA-mediated post-transcriptional regulation as a key mechanism linking molecular alterations to cellular and network-level deficits in SCZ.

cell biology↗

bioRxiv2025

MrgD Receptor Modulates Neurotransmission in the Nigrostriatal Pathway

The Mas-related G protein-coupled receptor D (MrgD) is primarily known for its role in peripheral nociception and, more recently, as a receptor for alamandine, influencing cardiovascular function and exhibiting antidepressant-like effects. However, its function within the central nervous system, particularly in motor and reward-related circuits, remains largely unexplored. Here, we investigate the role of MrgD in the nigrostriatal pathway using proteomic approaches focused on post-translational modifications in MrgD-knockout (KO) mice. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses revealed significant alterations in synaptic vesicle-associated proteins, pointing to impaired neurotransmission in the nigrostriatal system of KO mice. These molecular findings were supported by neurotransmitter quantification and functional assays demonstrating impaired synaptic vesicle exocytosis. Pharmacodynamic analyses showed that MrgD modulates synaptic exocytosis in an agonist-selective manner, being responsive to alamandine but not {beta}-alanine. Furthermore, behavioral analyses revealed increased locomotor activity and compulsive-like behavior in MrgD-deficient mice, without impairments in short- or long-term memory. Together, these findings uncover a new role for MrgD beyond its involvement in nociception, highlighting this receptor as a potential therapeutic target for neurological disorders involving motor hyperactivity and compulsivity.

neuroscience↗

bioRxiv2024

Adipogenin Dictates Adipose Tissue Expansion by Facilitating the Assembly of a Dodecameric Seipin Complex

Adipogenin (Adig) is an evolutionarily conserved microprotein and is highly expressed in adipose tissues and testis. Here, we identify Adig as a critical regulator for lipid droplet formation in adipocytes. We determine that Adig interacts directly with seipin, leading to the formation of a rigid complex. We solve the structure of the seipin/Adig complex by Cryo-EM at 2.98[A] overall resolution. Surprisingly, seipin can form two unique oligomers, undecamers and dodecamers. Adig selectively binds to the dodecameric seipin complex. We further find that Adig promotes seipin assembly by stabilizing and bridging adjacent seipin subunits. Functionally, Adig plays a key role in generating lipid droplets in adipocytes. In mice, inducible overexpression of Adig in adipocytes substantially increases fat mass, with enlarged lipid droplets. It also elevates thermogenesis during cold exposure. In contrast, inducible adipocyte-specific Adig knockout mice manifest aberrant lipid droplet formation in brown adipose tissues and impaired cold tolerance.

cell biology↗

bioRxiv2024

Angiotensin-(1-5) is a Potent Endogenous Angiotensin AT2-Receptor Agonist

BackgroundThe renin-angiotensin system involves many more enzymes, receptors and biologically active peptides than originally thought. With this study, we investigated whether angiotensin-(1-5) [Ang-(1-5)], a 5-amino acid fragment of angiotensin II, has biological activity, and through which receptor it elicits effects. MethodsThe effect of Ang-(1-5) (1{micro}M) on nitric oxide release was measured by DAF-FM staining in human aortic endothelial cells (HAEC), or Chinese Hamster Ovary (CHO) cells stably transfected with the angiotensin AT2-receptor (AT2R) or the receptor Mas. A potential vasodilatory effect of Ang-(1-5) was tested in mouse mesenteric and human renal arteries by wire myography; the effect on blood pressure was evaluated in normotensive C57BL/6 mice by Millar catheter. These experiments were performed in the presence or absence of a range of antagonists or inhibitors or in AT2R-knockout mice. Binding of Ang-(1-5) to the AT2R was confirmed and the preferred conformations determined by in silico docking simulations. The signaling network of Ang-(1-5) was mapped by quantitative phosphoproteomics. ResultsKey findings included: (1) Ang-(1-5) induced activation of eNOS by changes in phosphorylation at Ser1177eNOS and Tyr657eNOS and thereby (2) increased NO release from HAEC and AT2R-transfected CHO cells, but not from Mas-transfected or non-transfected CHO cells. (3) Ang-(1-5) induced relaxation of preconstricted mouse mesenteric and human renal arteries and (4) lowered blood pressure in normotensive mice - effects which were respectively absent in arteries from AT2R-KO or in PD123319-treated mice and which were more potent than effects of the established AT2R-agonist C21. (5) According to in silico modelling, Ang-(1-5) binds to the AT2R in two preferred conformations, one differing substantially from where the first five amino acids within angiotensin II bind to the AT2R. (6) Ang-(1-5) modifies signaling pathways in a protective RAS-typical way and with relevance for endothelial cell physiology and disease. ConclusionsAng-(1-5) is a potent, endogenous AT2R-agonist.

pharmacology and toxicology↗

bioRxiv2023

Off-targets of BRAF inhibitors disrupt endothelial signaling and differentially affect vascular barrier function

Targeted therapies against mutant BRAF are effectively used in combination with MEK inhibitors (MEKi) to treat advanced melanoma. However, treatment success is affected by resistance and adverse events (AEs). Approved BRAF inhibitors (BRAFi) show high levels of target promiscuity, which can contribute to these effects. Blood vessels are in direct contact with high plasma concentrations of BRAFi, but effects of the inhibitors in this cell type are unknown. Hence, we aimed to characterize responses to approved BRAFi for melanoma in the vascular endothelium. We showed that all clinically approved BRAFi induced a paradoxical activation of endothelial MAPK signaling. Moreover, phosphoproteomics revealed distinct sets of off-targets per inhibitor. Endothelial barrier function and junction integrity were impaired upon treatment with Vemurafenib and the next-generation dimerization inhibitor PLX8394, but not with Dabrafenib or Encorafenib. Together, these findings provide insights on the surprisingly distinct side effects of BRAFi on endothelial signaling and functionality. Better understanding of off-target effects could help to identify molecular mechanisms behind AEs and guide the continued development of therapies for BRAF-mutant melanoma.

cell biology↗

bioRxiv2022

A long-acting GDF15 analog causes robust, sustained weight loss and reduction of food intake in an obese non-human primate model

GDF15 is a circulating polypeptide associated with cellular stress, and recently linked to metabolic adaptation. GDF15 has a half-life of approximately 3 hours in and acts at the GFRAL receptor selectively expressed in the area postrema. To characterize the effects of sustained GFRAL agonism on food intake (FI) and body weight (BW), we developed a half-life extended analog of GDF15 (Compound H; CpdH) suitable for reduced dosing frequency and tested its effects in obese cynomolgus monkeys. Animals were treated once weekly for 12 weeks with 0.048, 0.16, or 1.6 mg/Kg of CpdH or with 0.02 mg/Kg of the long-acting GLP-1 analog dulaglutide as a positive control. FI was measured daily and BW was measured biweekly. Mechanism-based longitudinal exposure-response (E-R) modeling was performed to characterize the effects of CpdH and dulaglutide on FI and BW. The integrated novel model accounts for both acute, exposure-dependent effects of treatments to reduce FI and the compensatory changes in energy expenditure (EE) and FI that occur over time in response to weight loss. CpdH had approximately linear, dose-proportional pharmacokinetics with a half-life of {approx}8 days and treatment with CpdH led to dose- and exposure-dependent reductions in FI and BW. The 1.6 mg/Kg CpdH dose reduced mean FI by 57.5% at 1 week and provided sustained FI reductions of 31.5% from weeks 9-12, leading to a peak reduction in BW of 16{+/-}5%. Dulaglutide had more modest effects on FI (reductions between 15-40%) and peak BW loss was 3.8{+/-}4.0%. Longitudinal modeling of both the FI and BW profiles suggested reductions in BW observed with both CpdH and dulaglutide were fully explained by the exposure-dependent reductions in FI without any increase in EE. Upon verification of pharmacokinetic/pharmacodynamic relationship established in monkeys and human for dulaglutide, we predicted that CpdH could reach double digit BW loss in human. In summary, treatment with a long-acting GDF15 analog led to sustained dose- and exposure-dependent reductions in food intake in a monkey model of obesity and holds potential for effective clinical obesity pharmacotherapy. Significance StatementGDF15 activation of GFRAL receptors in the hindbrain controls food intake and body weight. Here we describe the effect and durability of a circulating half-life extended analog of GDF15 (Compound H) on food intake and body weight loss in a spontaneously obese cynomolgus monkey model. Inclusion of a translational treatment arm with a weight loss agent, dulaglutide, permitted pharmacokinetic/pharmacodynamic modeling and comparison of both GDF15 and GLP-1 based weight loss mechanisms, and development of an allometric scaling based mathematical model to estimate the efficacy of Compound H in human obese subjects.

pharmacology and toxicology↗

bioRxiv2021

The phosphoproteome of rice leaves responds to water and nitrogen supply

The scarcity of freshwater is an increasing concern in flood-irrigated rice, whilst excessive use of nitrogen fertilizers is both costly and contributes to environmental pollution. To co-ordinate growth adaptation under prolonged exposure to limited water or excess nitrogen supply, plants have processes for signalling and regulation of metabolic processes. There is limited information on the involvement of one of the most important post-translational modifications (PTMs), protein phosphorylation, on plant adaptation to long-term changes in resource supply. Oryza sativa cv. Nipponbare was grown under two regimes of nitrogen from the time of germination to final harvest. Twenty-five days after germination, water was withheld from half the pots in each nitrogen treatment and low water supply continued for an additional 26 days, while the remaining pots were well watered. Leaves from all four groups of plants were harvested after 51 days in order to test whether phosphorylation of leaf proteins responded to prior abiotic events. The dominant impact of these resources is exerted in leaves, where PTMs have been predicted to occur. Proteins were extracted and phosphopeptides were analysed by nanoLC-MS/MS analysis, coupled with label-free quantitation. Water and nitrogen regimes triggered extensive changes in phosphorylation of proteins involved in membrane transport, such as the aquaporin OsPIP2-6, a water channel protein. Our study reveals phosphorylation of several peptides belonging to proteins involved in RNA-processing and carbohydrate metabolism, suggesting that phosphorylation events regulate the signalling cascades that are required to optimize plant response to resource supply.

plant biology↗

bioRxiv2020

Increasing proteome coverage using cysteine-specific DIA Mass spectrometry - Cys-DIA

We report a Data Independent Analysis (DIA) mass spectrometric method for quantitative proteomics based on cysteine-containing peptides derived by tryptic digestion from biological samples (termed Cys-DIA). This technology significantly increases the coverage of the identifiable and quantifiable proteome in DIA/SWATH analyses by >35% compared to the conventional DIA/SWATH methods. The strategy was applied here to illustrate the quantitative difference in the proteome between isogenic human cancer cell lines with different metastatic capabilities.

biochemistry↗