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Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3′ region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.

The outbreak of a novel coronavirus (SARS-CoV-2) in 2019 led to a worldwide pandemic, which remains an integral part of our lives to this day. Coronavirus disease (COVID-19) is a flu like condition, often accompanied by high fever and respiratory distress. In some cases, conjointly with other co-morbidities, COVID-19 can become severe, leading to lung arrest and even death. Although well-known from a clinical standpoint, the mechanistic understanding of lethal COVID-19 is still rudimentary. Studying the pathology and changes on a molecular level associated with the resulting COVID-19 disease is impeded by the highly infectious nature of the virus and the concomitant sampling challenges. We were able to procure COVID-19 post-mortem lung tissue specimens by our collaboration with the BSL-3 laboratory of the Biobanking and BioMolecular resources Research Infrastructure Austria which we subjected to state-of-the-art quantitative proteomic analysis to better understand the pulmonary manifestations of lethal COVID-19. Lung tissue samples from age-matched non-COVID-19 patients who died within the same period were used as controls. Samples were subjected to parallel accumulation–serial fragmentation combined with data-independent acquisition (diaPASEF) on a timsTOF Pro and obtained raw data was processed using DIA-NN software. Here we report that terminal COVID-19 patients display an increase in inflammation, acute immune response and blood clot formation (with concomitant triggering of fibrinolysis). Furthermore, we describe that COVID-19 diseased lungs undergo severe extracellular matrix restructuring, which was corroborated on the histopathological level. However, although undergoing an injury, diseased lungs seem to have impaired proliferative and tissue repair signalling, with several key kinase-mediated signalling pathways being less active. This might provide a mechanistic link to post-acute sequelae of COVID-19 (PASC; “Long COVID”). Overall, we emphasize the importance of histopathological patient stratification when interpreting molecular COVID-19 data.

Background In addition to known allergens, other proteins in pollen can aid the development of an immune response in allergic individuals. The contribution of the “unknown” protein allergens is apparent in phylogenetically related species where, despite of high homology of the lead allergens, the degree of allergenic potential can vary greatly. The aim of this study was to identify other potentially allergenic proteins in pollen of three common and highly related allergenic tree species: birch (Betula pendula), hazel (Corylus avellana) and alder (Alnus glutinosa). Methods For that purpose, we carried out a comprehensive, comparative proteomic screening of the pollen from the three species. In order to maximize protein recovery and coverage, different protein extraction and isolation strategies during sample preparation were employed. Results As a result, we report 2500–3000 identified proteins per each of the pollen species. Identified proteins were further used for a number of annotation steps, providing insight into differential distribution of peptidases, peptidase inhibitors and other potential allergenic proteins across the three species. Moreover, we carried out functional enrichment analyses that, interestingly, corroborated high species similarity in spite of their relatively distinct protein profiles. Conclusion We provide to our knowledge first insight into proteomes of two very important allergenic pollen types, hazel and alder, where not even transcriptomics data are available, and compared them to birch. Datasets from this study can be readily used as protein databases and as such serve as basis for further functional studies., This study aims to identify and annotate novel potentially allergenic proteins in the three highly related pollen species: birch, hazel, and alder. Immunoblotting of pollen protein extracts with serum from allergenic patients corroborates differential allergenic potential despite of the high similarity of the three species. Comprehensive proteomics analysis provides the first available protein database of pollens of alder and hazel and enables thorough annotation of proteases and allergens as well as comparative functional analysis of expressed proteins across the three pollens. Abbreviations: LC‐MS, liquid chromatography‐mass spectrometry; SDS‐PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Cholangiocarcinoma (CCA) are characterized by their desmoplastic and hypervascularized tumor microenvironment (TME), which is mainly composed of tumor cells and cancer-associated fibroblasts (CAFs). CAFs play a pivotal role in general and CCA tumor progression, angiogenesis, metastasis, and the development of treatment resistance. To our knowledge, no continuous human in vivo-like co-culture model is available for research. Therefore, we aimed to establish a new model system (called MUG CCArly) that mimics the desmoplastic microenvironment typically seen in CCA. Proteomic data comparing the new CCA tumor cell line with our co-culture tumor model (CCTM) indicated a higher gene expression correlation of the CCTM with physiological CCA characteristics. A pro-angiogenic TME that is typically observed in CCA could also be better simulated in the CCTM group. Further analysis of secreted proteins revealed CAFs to be the main source of these angiogenic factors. Our CCTM MUG CCArly represents a new, reproducible, and easy-to-handle 3D CCA model for preclinical studies focusing on CCA-stromal crosstalk, tumor angiogenesis, and invasion, as well as the immunosuppressive microenvironment and the involvement of CAFs in the way that drug resistance develops.

Non-alcoholic fatty liver disease is a pathology with a hard-to-detect onset and is estimated to be present in a quarter of the adult human population. To improve our understanding of the development of non-alcoholic fatty liver disease, we treated a human hepatoma cell line model, HepG2, with increasing concentrations of common fatty acids, namely myristic, palmitic and oleic acid. To reproduce more physiologically representative conditions, we also included combinations of these fatty acids and monitored the cellular response with an in-depth proteomics approach and imaging techniques. The two saturated fatty acids initially presented a similar phenotype of a dose-dependent decrease in growth rates and impaired lipid droplet formation. Detailed analysis revealed that the drop in the growth rates was due to delayed cell-cycle progression following myristic acid treatment, whereas palmitic acid led to cellular apoptosis. In contrast, oleic acid, as well as saturated fatty acid mixtures with oleic acid, led to a dose-dependent increase in lipid droplet volume without adverse impacts on cell growth. Comparing the effects of harmful single-fatty-acid treatments and the well-tolerated fatty acid mixes on the cellular proteome, we were able to differentiate between fatty-acid-specific cellular responses and likely common lipotoxic denominators.

Background: Atrial fibrillation (AF), the most common sustained arrhythmia, is associated with increased morbidity, mortality, and health care costs. AF develops over many years and is often related to substantial atrial structural and electrophysiological remodeling. AF may lack symptoms at onset, and atrial biopsy samples are generally obtained in subjects with advanced disease, so it is difficult to study earlier stage pathophysiology in humans. Methods: Here, we characterized comprehensively the transcriptomic (miRNA-seq and mRNA-seq) changes in the left atria of 2 robust canine AF models after 1 week of electrically maintained AF, without or with ventricular rate control via atrioventricular node-ablation/ventricular pacing. Results: Our RNA-sequencing experiments identified thousands of genes that are differentially expressed, including a majority that have never before been implicated in AF. Gene set enrichment analyses highlighted known (eg, extracellular matrix structure organization) but also many novel pathways (eg, muscle structure development, striated muscle cell differentiation) that may play a role in tissue remodeling and cellular trans-differentiation. Of interest, we found dysregulation of a cluster of noncoding RNAs, including many microRNAs but also the MEG3 long noncoding RNA orthologue, located in the syntenic region of the imprinted human DLK1-DIO3 locus. Interestingly (in the light of other recent observations), our analysis identified gene targets of differentially expressed microRNAs at the DLK1-DIO3 locus implicating glutamate signaling in AF pathophysiology. Conclusions: Our results capture molecular events that occur at an early stage of disease development using well-characterized animal models and may, therefore, inform future studies that aim to further dissect the causes of AF in humans.

BackgroundAtrial fibrillation (AF), the most common sustained arrhythmia, is associated with increased morbidity, mortality, and health-care costs. AF develops over many years and is often related to substantial atrial structural and electrophysiological remodeling. AF may lack symptoms at onset and atrial biopsy samples are generally obtained in subjects with advanced disease, so it is difficult to study earlier-stage pathophysiology in humans.MethodsHere, we characterized comprehensively the transcriptomic (miRNAseq and mRNAseq) changes in the left atria of two robust canine AF-models after one week of electrically-maintained AF, without or with ventricular rate-control via atrioventricular node-ablation/ventricular pacing.ResultsOur RNA-sequencing experiments identified thousands of genes that are differentially expressed, including a majority that have never before been implicated in AF. Gene-set enrichment analyses highlighted known (e.g. extracellular matrix structure organization) but also many novel pathways (e.g. muscle structure development, striated muscle cell differentiation) that may play a role in tissue remodeling and/or cellular transdifferentiation. Of interest, we found dysregulation of a cluster of non-coding RNAs, including many microRNAs but also theMEG3long non-coding RNA orthologue, located in the syntenic region of the imprinted humanDLK1-DIO3locus. Interestingly (in the light of other recent observations), our analysis identified gene-targets of differentially expressed microRNAs at theDLK1-DIO3locus implicating glutamate signaling in AF pathophysiology.ConclusionsOur results capture molecular events that occur at an early stage of disease development using well-characterized animal models, and may therefore inform future studies that aim to further dissect the causes of AF in humans.

Heart failure with preserved ejection fraction (HFpEF) is a major health problem without effective therapies. This study assessed the effects of histone deacetylase (HDAC) inhibition on cardiopulmonary structure, function, and metabolism in a large mammalian (feline) model of pressure overload that recapitulates features of diastolic dysfunction common to human HFpEF. Male domestic short hair felines (n=31, aged 2 mo), underwent a sham procedure (n=10) or loose aortic banding (n=21), resulting in slow-progressive pressure overload during growth. Two months post-banding, animals were treated daily with suberoylanilide hydroxamic acid (b+SAHA, 10 mg/kg, n=8), a Food and Drug Administration (FDA)-approved pan-HDAC inhibitor, or vehicle (b+veh, n=8) for 2 months. Echocardiography at 4-months post-banding revealed that b+SAHA animals had significantly reduced left ventricular hypertrophy (LVH) (P < 0.0001) and left atrium size (P < 0.0001) vs b+veh animals. Left ventricular end-diastolic pressure and mean pulmonary arterial pressure were significantly reduced in b+SAHA (P < 0.01) vs b+veh. SAHA increased myofibril relaxation ex vivo, which correlated with in vivo improvements of LV relaxation. Furthermore, SAHA treatment preserved lung structure, compliance, blood oxygenation, and reduced perivascular fluid cuffs around extra-alveolar vessels, suggesting attenuated alveolar-capillary stress failure. Acetylation proteomics revealed that SAHA altered lysine acetylation of mitochondrial metabolic enzymes. HDAC inhibition reduces LVH, LV diastolic dysfunction, and atrial and pulmonary remodeling in a feline model of diastolic dysfunction. These results suggest that acetylation defects in hypertrophic stress can be reversed by HDAC inhibitors, with implications for improving cardiac structure and function in patients.

Background Cardiac troponins are often elevated in patients with skeletal muscle disease who have no evidence of cardiac disease. Objectives The goal of this study was to characterize cardiac troponin concentrations in patients with myopathies and derive insights regarding the source of elevated troponin T measurements. Methods Cardiac troponin T (cTnT) and cardiac troponin I (cTnI) concentrations were determined by using high sensitivity assays in 74 patients with hereditary and acquired skeletal myopathies. Patients underwent comprehensive cardiac evaluation, including 12-lead electrocardiogram, 24-h electrocardiogram, cardiac magnetic resonance imaging, and coronary artery computed tomography. cTnT and cTnI protein expression was determined in skeletal muscle samples of 9 patients and in control tissues derived from autopsy using antibodies that are used in commercial assays. Relevant Western blot bands were subjected to liquid chromatography tandem mass spectrometry for protein identification. Results Levels of cTnT (median: 24 ng/l; interquartile range: 11 to 54 ng/l) were elevated (>14 ng/l) in 68.9% of patients; cTnI was elevated (>26 ng/l) in 4.1% of patients. Serum cTnT levels significantly correlated with creatine kinase and myoglobin (r = 0.679 and 0.786, respectively; both p Conclusions Measured cTnT concentrations were chronically elevated in the majority of patients with skeletal myopathies, whereas cTnI elevation was rare. Our data indicate that cross-reaction of the cTnT immunoassay with skeletal muscle troponin isoforms was the likely cause.

Technical advances including liquid chromatography–tandem mass spectrometry and its data analysis enable detailed proteomic analysis of the nasal mucus. Alterations of the nasal mucus proteome may provoke substantial changes of the nasal physiology and have already been associated with rhinologic diseases such as allergic rhinitis. This study was conducted as a pilot study to map the olfactory cleft proteome using current techniques for proteomic analysis. Furthermore, we aimed to investigate proteomic changes as potential biomarkers in patients suffering from idiopathic and postinfectious olfactory disorders compared to healthy controls. Seven patients with idiopathic hyposmia and anosmia, seven patients with postinfectious hyposmia and anosmia and seven healthy controls were included in this study. In total, 1117 different proteins were detected in at least five patients in at least one group. Results of this study did not reveal significant differences regarding the proteomic composition of the olfactory cleft mucus between patients versus healthy controls. Among proteins involved in olfactory perception the G protein family was detected but also found unchanged between groups. Investigation of protein composition by liquid chromatography–tandem mass spectrometry enabled us to perform an in–depth analysis of the olfactory cleft mucus proteome regarding the diversity of different proteins in individual patients. However untargeted proteomics of the olfactory cleft mucus may not be an applicable approach to develop biomarkers for olfactory disorders. Targeted analyses of distinct proteins known to be involved in olfactory perception but not detected by our approach, e.g. odorant binding proteins, may provide more information regarding pathophysiology of olfactory diseases.

Myristic acid, the 14-carbon saturated fatty acid (C14:0), is associated to an increased cardiovascular disease risk. Since it is found in low concentration in cells, its specific properties have not been fully analyzed. The aim of this study was to explore the cell response to this fatty acid to help explaining clinical findings on the relationship between C14:0 and cardiovascular disease. The human liver HepG2 cell line was used to investigate the hepatic response to C14:0 in a combined proteomic and secretomic approach. A total of 47 intracellular and 32 secreted proteins were deregulated after treatments with different concentrations of C14:0. Data are available via ProteomeXchange ( PXD007902 ). In addition, C14:0 treatment of primary murine hepatocytes confirmed that C14:0 induces lipid droplet accumulation and elevates perilipin-2 levels. Functional enrichment analysis revealed that C14:0 modulates lipid droplet formation and cytoskeleton organization, induce ER stress, changes in exosome and extracellular miRNA sorting in HepG2cells. Our data provide for the first time a proteomic profiling of the effects of C14:0 in human hepatoma cells and contribute to the elucidation of molecular mechanisms through which this fatty acid may cause adverse health effects. Biological significance Myristic acid is correlated with an increase in plasma cholesterol and mortality due to cardiovascular diseases. This study is the first example of an integration of proteomic and secretomic analysis of HepG2 cells to investigate the specific properties and functional roles of myristic acid on hepatic cells. Our analyses will lead to a better understanding of the myristic acid induced effects and can elicit new diagnostic and treatment strategies based on altered proteins.

Chemically modified trypsin is a standard reagent in proteomics experiments but is usually not considered in database searches. Modification of trypsin is supposed to protect the protease against autolysis and the resulting loss of activity. Here, we show that modified trypsin is still subject to self-digestion, and, as a result, modified trypsin-derived peptides are present in standard digests. We depict that these peptides commonly lead to false-positive assignments even if native trypsin is considered in the database. Moreover, we present an easily implementable method to include modified trypsin in the database search with a minimal increase in search time and search space while efficiently avoiding these false-positive hits.