Rbp95 binds to 25S rRNA helix H95 and cooperates with the Npa1 complex during early pre-60S particle maturation.

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.

Automated Mass Photometry of Adeno-Associated Virus Vectors from Crude Cell Extracts.

Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires minimal sample volumes between 5-10 µL, which makes it a promising candidate over orthogonal techniques such as analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (Cryo-TEM) or charge-detection mass spectrometry (CDMS). However, these methods are limited in their application to purified samples only. Here we developed a purification step based on single-domain monospecific antibody fragments immobilised on either a poly(styrene-divinylbenzene) resin or on magnetic beads prior to MP analysis that allows the quantification of empty, partially filled, full and overfull AAV vectors in crude cell extracts. This is aimed at identifying potentially promising harvest conditions that yield large numbers of filled AAV vectors during the early stages of the viral vector development platform, e.g., the type of transfection reagent used. Furthermore, we provide a direct comparison of the automated and manual handling of the mass photometer with respect to the quantities of AAV subspecies, molar mass of the capsid and payload, and highlight the differences between the "buffer-free" sample measurement and the "buffer-dilution" mode. In addition, we provide information on which candidates to use for calibration and demonstrate the limitations of the mass photometer with respect to the estimation of the capsid titer.

Human Sterols Are Overproduced, Stored and Excreted in Yeasts.

Sterols exert a profound influence on numerous cellular processes, playing a crucial role in both health and disease. However, comprehending the effects of sterol dysfunction on cellular physiology is challenging. Consequently, numerous processes affected by impaired sterol biosynthesis still elude our complete understanding. In this study, we made use of yeast strains that produce cholesterol instead of ergosterol and investigated the cellular response mechanisms on the transcriptome as well as the lipid level. The exchange of ergosterol for cholesterol caused the downregulation of phosphatidylethanolamine and phosphatidylserine and upregulation of phosphatidylinositol and phosphatidylcholine biosynthesis. Additionally, a shift towards polyunsaturated fatty acids was observed. While the sphingolipid levels dropped, the total amounts of sterols and triacylglycerol increased, which resulted in 1.7-fold enlarged lipid droplets in cholesterol-producing yeast cells. In addition to internal storage, cholesterol and its precursors were excreted into the culture supernatant, most likely by the action of ABC transporters Snq2, Pdr12 and Pdr15. Overall, our results demonstrate that, similarly to mammalian cells, the production of non-native sterols and sterol precursors causes lipotoxicity in K. phaffii, mainly due to upregulated sterol biosynthesis, and they highlight the different survival and stress response mechanisms on multiple, integrative levels.

Metabolic changes and propensity for inflammation, fibrosis, and cancer in livers of mice lacking lysosomal acid lipase.

Lysosomal acid lipase (LAL) is the sole lysosomal enzyme responsible for the degradation of cholesteryl esters and triacylglycerols at acidic pH. Impaired LAL activity leads to LAL deficiency (LAL-D), a severe and fatal disease characterized by ectopic lysosomal lipid accumulation. Reduced LAL activity also contributes to the development and progression of non-alcoholic fatty liver disease (NAFLD). To advance our understanding of LAL-related liver pathologies, we performed comprehensive proteomic profiling of livers from mice with systemic genetic loss of LAL (Lal-/-) and from mice with hepatocyte-specific LAL-D (hepLal-/-). Lal-/- mice exhibited drastic proteome alterations, including dysregulation of multiple proteins related to metabolism, inflammation, liver fibrosis, and cancer. Global loss of LAL activity impaired both acidic and neutral lipase activities and resulted in hepatic lipid accumulation, indicating a complete metabolic shift in Lal-/- livers. Hepatic inflammation and immune cell infiltration were evident, with numerous upregulated inflammation-related gene ontology biological process terms. In contrast, both young and mature hepLal-/- mice displayed only minor changes in the liver proteome, suggesting that loss of LAL solely in hepatocytes does not phenocopy metabolic alterations observed in mice globally lacking LAL. These findings provide valuable insights into the mechanisms underlying liver dysfunction in LAL-D and may help in understanding why decreased LAL activity contributes to NAFLD. Our study highlights the importance of LAL in maintaining liver homeostasis and demonstrates the drastic consequences of its global deficiency on the liver proteome and liver function.

Redox-driven cardioprotective effects of sodium-glucose co-transporter-2 inhibitors: comparative review.

Sodium-glucose co-transporter-2 inhibitors are used in the treatment of diabetes but are also emerging as cardioprotective agents in heart diseases even in the absence of type 2 diabetes. In this paper, upon providing a short overview of common pathophysiological features of diabetes, we review the clinically reported cardio- and nephroprotective potential of sodium-glucose co-transporter-2 inhibitors currently available on the market, including Dapagliflozin, Canagliflozin, and Empagliflozin. To that end, we summarize findings of clinical trials that have initially drawn attention to the drugs' organ-protective potential, before providing an overview of their proposed mechanism of action. Since we particularly expect that their antioxidative properties will broaden the application of gliflozins from therapeutic to preventive care, special emphasis was put on this aspect.

Complementary omics strategies to dissect p53 signaling networks under nutrient stress.

Signaling trough p53is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Adipose Triglyceride Lipase Loss Promotes a Metabolic Switch in A549 Non-Small Cell Lung Cancer Cell Spheroids.

Cancer cells undergo complex metabolic adaptations to survive and thrive in challenging environments. This is particularly prominent for solid tumors, where cells in the core of the tumor are under severe hypoxia and nutrient deprivation. However, such conditions are often not recapitulated in the typical 2D in vitro cancer models, where oxygen as well as nutrient exposure is quite uniform. The aim of this study was to investigate the role of a key neutral lipid hydrolase, namely adipose triglyceride lipase (ATGL), in cancer cells that are exposed to more tumor-like conditions. To that end, we cultured lung cancer cells lacking ATGL as multicellular spheroids in 3D and subjected them to comprehensive proteomics analysis and metabolic phenotyping. Proteomics data are available via ProteomeXchange with identifier PXD021105. As a result, we report that loss of ATGL enhanced growth of spheroids and facilitated their adaptation to hypoxia, by increasing the influx of glucose and endorsing a pro-Warburg effect. This was followed by changes in lipid metabolism and an increase in protein production. Interestingly, the observed phenotype was also recapitulated in an even more "in vivo like" setup, when cancer spheroids were grown on chick chorioallantoic membrane, but not when cells were cultured as a 2D monolayer. In addition, we demonstrate that according to the publicly available cancer databases, an inverse relation between ATGL expression and higher glucose dependence can be observed. In conclusion, we provide indications that ATGL is involved in regulation of glucose metabolism of cancer cells when grown in 3D (mimicking solid tumors) and as such could be an important factor of the treatment outcome for some cancer types. Finally, we also ratify the need for alternative cell culture models, as the majority of phenotypes observed in 3D and spheroids grown on chick chorioallantoic membrane were not observed in 2D cell culture.

Quantification of Empty, Partially Filled and Full Adeno-Associated Virus Vectors Using Mass Photometry.

Adeno-associated viruses (AAV) are one of the most commonly used vehicles in gene therapies for the treatment of rare diseases. During the AAV manufacturing process, particles with little or no genetic material are co-produced alongside the desired AAV capsid containing the transgene of interest. Because of the potential adverse health effects of these byproducts, they are considered impurities and need to be monitored carefully. To date, analytical ultracentrifugation (AUC), transmission electron microscopy (TEM) and charge-detection mass spectrometry (CDMS) are used to quantify these subspecies. However, they are associated with long turnaround times, low sample throughput and complex data analysis. Mass photometry (MP) is a fast and label-free orthogonal technique which is applicable to multiple serotypes without the adaption of method parameters. Furthermore, it can be operated with capsid titers as low as 8 × 1010 cp mL-1 with a CV < 5% using just 10 µL total sample volume. Here we demonstrate that mass photometry can be used as an orthogonal method to AUC to accurately quantify the proportions of empty, partially filled, full and overfull particles in AAV samples, especially in cases where ion-exchange chromatography yields no separation of the populations. In addition, it can be used to confirm the molar mass of the packaged genomic material in filled AAV particles.

Proteomic profiling of end-stage COVID-19 lung biopsies.

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.

Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags.

Vibrio natriegens is the fastest growing organism identified so far. The minimum doubling time of only 9.4 min, the ability to utilize over 60 different carbon sources and its non-pathogenic properties make it an interesting alternative to E. coli as a new production host for recombinant proteins. We investigated the ability of the engineered V. natriegens strain, Vmax™ Express, to incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into recombinant proteins for NMR applications. AzF was incorporated into enhanced yellow fluorescent protein (EYFP) and MlaC, an intermembrane transport protein, by stop codon suppression. AzF incorporation into EYFP resulted in an improved suppression efficiency (SE) of up to 35.5 ± 0.8% and a protein titer of 26.7 ± 0.7 mg/L. The expression levels of MlaC-AzF even exceeded those of E. coli BL21 cells. For the recording of 1H-15N and 19F NMR spectra, EYFP-AzF was expressed and isotopically labeled in minimal medium and the newly introduced azido-group was used as coupling site for NMR sensitive 19F-tags. Our findings show that Vmax is a flexible expression host, suitable for the incorporation of ncAAs in recombinant proteins with the potential to surpass protein yields of E. coli. The presented method suggests the implementation of V. natriegens for expression of isotopically labeled proteins containing ncAAs, which can be chemically modified for the application in protein-observed 19F-NMR.

Spatially Resolved Activity-based Proteomic Profiles of the Murine Small Intestinal Lipases.

Despite the crucial function of the small intestine in nutrient uptake our understanding of the molecular events underlying the digestive function is still rudimentary. Recent studies demonstrated that enterocytes do not direct the entire dietary triacylglycerol toward immediate chylomicron synthesis. Especially after high-fat challenges, parts of the resynthesized triacylglycerol are packaged into cytosolic lipid droplets for transient storage in the endothelial layer of the small intestine. The reason for this temporary storage of triacylglycerol is not completely understood. To utilize lipids from cytosolic lipid droplets for chylomicron synthesis in the endoplasmic reticulum, stored triacylglycerol has to be hydrolyzed either by cytosolic lipolysis or lipophagy. Interestingly, triacylglycerol storage and chylomicron secretion rates are unevenly distributed along the small intestine, with the proximal jejunum exhibiting the highest intermittent storage capacity. We hypothesize that correlating hydrolytic enzyme activities with the reported distribution of triacylglycerol storage and chylomicron secretion in different sections of the small intestine is a promising strategy to determine key enzymes in triacylglycerol remobilization. We employed a serine hydrolase specific activity-based labeling approach in combination with quantitative proteomics to identify and rank hydrolases based on their relative activity in 11 sections of the small intestine. Moreover, we identified several clusters of enzymes showing similar activity distribution along the small intestine. Merging our activity-based results with substrate specificity and subcellular localization known from previous studies, carboxylesterase 2e and arylacetamide deacetylase emerge as promising candidates for triacylglycerol mobilization from cytosolic lipid droplets in enterocytes.

Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors.

Ion-exchange chromatography coupled to light scattering detectors represents a fast and simple analytical method for the assessment of multiple critical quality attributes (CQA) in one single measurement. The determination of CQAs play a crucial role in Adeno-Associated Virus (AAV)-based gene therapies and their applications in humans. Today, several different analytical techniques, including size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), qPCR or ELISA, are commonly used to characterize the gene therapy product regarding capsid titer, packaging efficiency, vector genome integrity, aggregation content and other process-related impurities. However, no universal method for the simultaneous determination of multiple CQAs is currently available. Here, we present a novel robust ion-exchange chromatography method coupled to multi-angle light scattering detectors (IEC-MALS) for the comprehensive characterization of empty and filled AAVs concerning capsid titer, full-to-total ratio, absolute molar mass of the protein and nucleic acid, and the size and polydispersity without baseline-separation of both species prior to data analysis. We demonstrate that the developed IEC-MALS assay is applicable to different serotypes and can be used as an orthogonal method to other established analytical techniques.

Hepatocyte Proteome Alterations Induced by Individual and Combinations of Common Free Fatty Acids.

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.

The Crystal Structure of Mouse Ces2c, a Potential Ortholog of Human CES2, Shows Structural Similarities in Substrate Regulation and Product Release to Human CES1.

Members of the carboxylesterase 2 (Ces2/CES2) family have been studied intensively with respect to their hydrolytic function on (pro)drugs, whereas their physiological role in lipid and energy metabolism has been realized only within the last few years. Humans have one CES2 gene which is highly expressed in liver, intestine, and kidney. Interestingly, eight homologous Ces2 (Ces2a to Ces2h) genes exist in mice and the individual roles of the corresponding proteins are incompletely understood. Mouse Ces2c (mCes2c) is suggested as potential ortholog of human CES2. Therefore, we aimed at its structural and biophysical characterization. Here, we present the first crystal structure of mCes2c to 2.12 Å resolution. The overall structure of mCes2c resembles that of the human CES1 (hCES1). The core domain adopts an α/ß hydrolase-fold with S230, E347, and H459 forming a catalytic triad. Access to the active site is restricted by the cap, the flexible lid, and the regulatory domain. The conserved gate (M417) and switch (F418) residues might have a function in product release similar as suggested for hCES1. Biophysical characterization confirms that mCes2c is a monomer in solution. Thus, this study broadens our understanding of the mammalian carboxylesterase family and assists in delineating the similarities and differences of the different family members.

Comparative proteomics of common allergenic tree pollens of birch, alder, and hazel.

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.

A possible role of gas-phase electrophoretic mobility molecular analysis (nES GEMMA) in extracellular vesicle research.

The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Platelet-derived extracellular vesicles (EVs)with/without additional size exclusion chromatographic (SEC) purification were subjected to nanoparticle tracking analysis (NTA) and gas-phase electrophoresis (nES GEMMA). The latter revealed presence of co-purified proteins, targetable via mass spectrometry (MS). MS also revealed that SEC did not influence EV protein content. To conclude, nES GEMMA is a valuable tool for quality control of EV-containing samples under native conditions allowing for detection of co-purified proteins from complex matrices.

Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema.

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options.

Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME.

Dedicated chaperones protect newly synthesized ribosomal proteins (r-proteins) from aggregation and accompany them on their way to assembly into nascent ribosomes. Currently, only nine of the ∼80 eukaryotic r-proteins are known to be guarded by such chaperones. In search of new dedicated r-protein chaperones, we performed a tandem-affinity purification based screen and looked for factors co-enriched with individual small subunit r-proteins. We report the identification of Nap1 and Tsr4 as direct binding partners of Rps6 and Rps2, respectively. Both factors promote the solubility of their r-protein clients in vitro. While Tsr4 is specific for Rps2, Nap1 has several interaction partners including Rps6 and two other r-proteins. Tsr4 binds co-translationally to the essential, eukaryote-specific N-terminal extension of Rps2, whereas Nap1 interacts with a large, mostly eukaryote-specific binding surface of Rps6. Mutation of the essential Tsr4 and deletion of the non-essential Nap1 both enhance the 40S synthesis defects of the corresponding r-protein mutants. Our findings highlight that the acquisition of eukaryote-specific domains in r-proteins was accompanied by the co-evolution of proteins specialized to protect these domains and emphasize the critical role of r-protein chaperones for the synthesis of eukaryotic ribosomes.

Proteomic Changes of Activated Hepatic Stellate Cells.

Hepatic stellate cells (HSC) are the major cellular drivers of liver fibrosis. Upon liver inflammation caused by a broad range of insults including non-alcoholic fatty liver, HSC transform from a quiescent into a proliferating, fibrotic phenotype. Although much is known about the pathophysiology of this process, exact cellular processes which occur in HSC and enable this transformation remain yet to be elucidated. In order to investigate this HSC transformation, we employed a simple, yet reliable model of HSC activation via an increase in growth medium serum concentration (serum activation). For that purpose, immortalized human LX-2 HSC were exposed to either 1% or 10% fetal bovine serum (FBS). Resulting quiescent (1% FBS) and activated (10% FBS) LX-2 cells were then subjected to in-depth mass spectrometry-based proteomics analysis as well as comprehensive phenotyping. Protein network analysis of activated LX-2 cells revealed an increase in the production of ribosomal proteins and proteins related to cell cycle control and migration, resulting in higher proliferation and faster migration phenotypes. Interestingly, we also observed a decrease in the expression of cholesterol and fatty acid biosynthesis proteins in accordance with a concomitant loss of cytosolic lipid droplets during activation. Overall, this work provides an update on HSC activation characteristics using contemporary proteomic and bioinformatic analyses and presents an accessible model for HSC activation. Data are available via ProteomeXchange with identifier PXD029121.

Mass Spectrometry-Based Redox and Protein Profiling of Failing Human Hearts.

Oxidative stress contributes to detrimental functional decline of the myocardium, leading to the impairment of the antioxidative defense, dysregulation of redox signaling, and protein damage. In order to precisely dissect the changes of the myocardial redox state correlated with oxidative stress and heart failure, we subjected left-ventricular tissue specimens collected from control or failing human hearts to comprehensive mass spectrometry-based redox and quantitative proteomics, as well as glutathione status analyses. As a result, we report that failing hearts have lower glutathione to glutathione disulfide ratios and increased oxidation of a number of different proteins, including constituents of the contractile machinery as well as glycolytic enzymes. Furthermore, quantitative proteomics of failing hearts revealed a higher abundance of proteins responsible for extracellular matrix remodeling and reduced abundance of several ion transporters, corroborating contractile impairment. Similar effects were recapitulated by an in vitro cell culture model under a controlled oxygen atmosphere. Together, this study provides to our knowledge the most comprehensive report integrating analyses of protein abundance and global and peptide-level redox state in end-stage failing human hearts as well as oxygen-dependent redox and global proteome profiles of cultured human cardiomyocytes.