Polyphosphate and tyrosine phosphorylation in the N-terminal domain of the human mitochondrial Lon protease disrupts its functions.

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.

How does ionizing radiation affect amyloidogenesis in plants?

PURPOSE: Ionizing radiation is a harsh environmental factor that could induce plant senescence. We hypothesized that radiation-related senescence remodels proteome, particularly by triggering the accumulation of prion-like proteins in plant tissues. The object of this study, pea (Pisum sativum L.), is an agriculturally important legume. Research on the functional importance of amyloidogenic proteins was never performed on this species. MATERIALS AND METHODS: Pea seeds were irradiated in the dose range 5-50 Gy of X-rays. Afterward, Fourier-transform infrared spectroscopy (FTIR) was used to investigate changes in the secondary structure of proteins in germinated 3-day-old seedlings. Specifically, we evaluated the ratio between the amide I and II peaks. Next, we performed protein staining with Congo red to compare the presence of amyloids in the samples. In parallel, we profiled the detergent-resistant proteome fraction by ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS). Differentially accumulated proteins were functionally analyzed in MapMan software, and the PLAAC tool was used to predict putative prion-like proteins. RESULTS: We showed a reduced germination rate but higher plant height and faster appearance of reproductive organs in the irradiated at dose of 50 Gy group compared with the control; furthermore, we demonstrated more ß-sheets and amyloid aggregates in the roots of stressed plants. We detected 531 proteins in detergent-resistant fraction extracted from roots, and 45 were annotated as putative prion-like proteins. Notably, 29 proteins were significantly differentially abundant between the irradiated and the control groups. These proteins belong to several functional categories: amino acid metabolism, carbohydrate metabolism, cytoskeleton organization, regulatory processes, protein biosynthesis, and RNA processing. Thus, the discovery proteomics provided deep data on novel aspects of plant stress biology. CONCLUSION: Our data hinted that protein accumulation stimulated seedlings' growth as well as accelerated ontogenesis and, eventually, senescence, primarily through translation and RNA processing. The increased abundance of primary metabolism-related proteins indicates more intensive metabolic processes triggered in germinating pea seeds upon X-ray exposure. The functional role of detected putative amyloidogenic proteins should be validated in overexpression or knockout follow-up studies.

Compartmentalization of galactan biosynthesis in mycobacteria.

Galactan polymer is a prominent component of the mycobacterial cell wall core. Its biogenesis starts at the cytoplasmic side of the plasma membrane by a build-up of the linker disaccharide [rhamnosyl (Rha) - N-acetyl-glucosaminyl (GlcNAc) phosphate] on the decaprenyl-phosphate carrier. This decaprenyl-P-P-GlcNAc-Rha intermediate is extended by two bifunctional galactosyl transferases, GlfT1 and GlfT2, and then it is translocated to the periplasmic space by an ABC transporter Wzm-Wzt. The cell wall core synthesis is finalized by the action of an array of arabinosyl transferases, mycolyl transferases, and ligases that catalyze an attachment of the arabinogalactan polymer to peptidoglycan through the linker region. Based on visualization of the GlfT2 enzyme fused with fluorescent tags it was proposed that galactan polymerization takes place in a specific compartment of the mycobacterial cell envelope, the intracellular membrane domain, representing pure plasma membrane free of cell wall components (previously denoted as the "PMf" domain), which localizes to the polar region of mycobacteria. In this work, we examined the activity of the galactan-producing cellular machine in the cell-wall containing cell envelope fraction and in the cell wall-free plasma membrane fraction prepared from Mycobacterium smegmatis by the enzyme assays using radioactively labeled substrate UDP-[14C]-galactose as a tracer. We found that despite a high abundance of GlfT2 in both of these fractions as confirmed by their thorough proteomic analyses, galactan is produced only in the reaction mixtures containing the cell wall components. Our findings open the discussion about the distribution of GlfT2 and the regulation of its activity in mycobacteria.

O-glycoprofiling of Serum Apolipoprotein C-III in Colorectal Cancer.

BACKGROUND: Aberrant glycosylation is a hallmark of cancer and thereby has an excellent potential for the discovery of novel biomarkers. Impairments in the glycan composition of lipoproteins impact their functional properties and can be associated with various diseases, including cancer. This research is still in its infancy; however, it can lead to the development of new diagnostic and disease stratification approaches as well as therapeutic strategies. Therefore, we aimed to evaluate anomalies in O-glycosylation of apolipoprotein C-III (apoC-III) in colorectal carcinoma (CRC) patients' sera, in comparison with sera from healthy individuals, and assess the disparities of O-glycoforms on apoC-III in CRC. METHODS: The choice of patients (n = 42) was based on the same tumor type (adenocarcinoma) and tumor size (T3), without or with inconsiderable lymph node infiltration. Patients with comorbidities were excluded from the study. The control healthy individuals (n = 40) were age- and sex-matched with patients. We used an approach based on the MALDI-TOF MS in linear positive ion mode, allowing simple analysis of O-glycosylation on intact apoC-III molecules in the serum samples directly, without the need for specific protein isolation. This approach enables relatively simple and high-throughput analysis. RESULTS: In CRC patients' sera samples, we observed significantly elevated apoC-III sialylation. Fully sialylated (disialylated) O-glycans had 1.26 times higher relative abundance in CRC samples compared to controls with a p-value of Mann-Whitney U test of 0.0021. CONCLUSIONS: We found altered O-glycosylation of apoC-III in the serum of CRC patients. However, it can be non-specific as it may be associated with another process such as ongoing inflammation. Therefore, to establish it as a potential novel non-invasive biomarker for CRC in suspected patients, further studies interrogating the changes in apoC-III O-glycosylation and the robustness of this biomarker need to be performed and evaluated.

Secretome analysis revealed that cell wall remodeling and starch catabolism underlie the early stages of somatic embryogenesis in Pinus nigra.

Somatic embryogenesis is an efficient mean for rapid micropropagation and preservation of the germplasm of valuable coniferous trees. Little is known about how the composition of secretome tracks down the level of embryogenic capacity. Unlike embryogenic tissue on solid medium, suspension cell cultures enable the study of extracellular proteins secreted into a liquid cultivation medium, avoiding contamination from destructured cells. Here, we present proteomic data of the secretome of Pinus nigra cell lines with contrasting embryogenic capacity, accounting for variability between genotypes. Our results showed that cell wall-related and carbohydrate-acting proteins were the most differentially accumulated. Peroxidases, extensin, α-amylase, plant basic secretory family protein (BSP), and basic secretory protease (S) were more abundant in the medium from the lines with high embryogenic capacity. In contrast, the medium from the low embryogenic capacity cell lines contained a higher amount of polygalacturonases, hothead protein, and expansin, which are generally associated with cell wall loosening or softening. These results corroborated the microscopic findings in cell lines with low embryogenic capacity-long suspensor cells without proper assembly. Furthermore, proteomic data were subsequently validated by peroxidase and α-amylase activity assays, and hence, we conclude that both tested enzyme activities can be considered potential markers of high embryogenic capacity.

Comparison of Fungal Thermophilic and Mesophilic Catalase-Peroxidases for Their Antioxidative Properties.

Catalase-peroxidases (KatGs) are unique bifunctional oxidoreductases that contain heme in their active centers allowing both the peroxidatic and catalatic reaction modes. These originally bacterial enzymes are broadly distributed among various fungi allowing them to cope with reactive oxygen species present in the environment or inside the cells. We used various biophysical, biochemical, and bioinformatics methods to investigate differences between catalase-peroxidases originating in thermophilic and mesophilic fungi from different habitats. Our results indicate that the architecture of the active center with a specific post-translational modification is highly similar in mesophilic and thermophilic KatG and also the peroxidatic acitivity with ABTS, guaiacol, and L-DOPA. However, only the thermophilic variant CthedisKatG reveals increased manganese peroxidase activity at elevated temperatures. The catalatic activity releasing molecular oxygen is comparable between CthedisKatG and mesophilic MagKatG1 over a broad temperature range. Two constructed point mutations in the active center were performed selectively blocking the formation of described post-translational modification in the active center. They exhibited a total loss of catalatic activity and changes in the peroxidatic activity. Our results indicate the capacity of bifunctional heme enzymes in the variable reactivity for potential biotech applications.

Subunit composition of mitochondrial dehydrogenase complexes in diplonemid flagellates.

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehydrogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH.

Alterations in the Glycan Composition of Serum Glycoproteins in Attention-Deficit Hyperactivity Disorder.

Changes in protein glycosylation are associated with most biological processes, and the importance of glycomic analysis in the research of disorders is constantly increasing, including in the neurodevelopmental field. We glycoprofiled sera in 10 children with attention-deficit hyperactivity disorder (ADHD) and 10 matching healthy controls for 3 types of samples: whole serum, sera after depletion of abundant proteins (albumin and IgG), and isolated IgG. The analytical methods used were a lectin-based glycoprotein microarray enabling high-throughput glycan analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as a standard method for the identification of glycan structures. For microarray analysis, the samples printed on microarray slides were incubated with biotinylated lectins and detected using the fluorescent conjugate of streptavidin by a microarray scanner. In the ADHD patient samples, we found increased antennary fucosylation, decreased di-/triantennary N-glycans with bisecting N-acetylglucosamine (GlcNAc), and decreased α2-3 sialylation. The results obtained by both independent methods were consistent. The study's sample size and design do not allow far-reaching conclusions to be drawn. In any case, there is a strong demand for a better and more comprehensive diagnosis of ADHD, and the obtained results emphasize that the presented approach brings new horizons to studying functional associations of glycan alterations in ADHD.

The Potential of Liquid Biopsy in Detection of Endometrial Cancer Biomarkers: A Pilot Study.

Endometrial cancer belongs to the most common gynecologic cancer types globally, with increasing incidence. There are numerous ways of classifying different cases. The most recent decade has brought advances in molecular classification, which show more accurate prognostic factors and the possibility of personalised adjuvant treatment. In addition, diagnostic approaches lag behind these advances, with methods causing patients discomfort while lacking the reproducibility of tissue sampling for biopsy. Minimally invasive liquid biopsies could therefore represent an alternative screening and diagnostic approach in patients with endometrial cancer. The method could potentially detect molecular changes in this cancer type and identify patients at early stages. In this pilot study, we tested such a detection method based on circulating tumour DNA isolated from the peripheral blood plasma of 21 Slovak endometrial cancer patients. We successfully detected oncomutations in the circulating DNA of every single patient, although the prognostic value of the detected mutations failed to offer certainty. Furthermore, we detected changes associated with clonal hematopoiesis, including DNMT3A mutations, which were present in the majority of circulating tumour DNA samples.

The Application of HPLC-FLD and NMR in the Monitoring of Therapy Efficacy in Alpha-Mannosidosis.

BACKGROUND: Alpha-mannosidosis is a rare lysosomal storage disorder, caused by decreased activity of α-D-mannosidase. This enzyme is involved in the hydrolysis of mannosidic linkages in N-linked oligosaccharides. Due to the mannosidase defect, undigested mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc) accumulating in cells are excreted in large quantities in urine. METHODS: In this work, we determined the levels of urinary mannose-rich oligosaccharides in a patient subjected to novel enzyme replacement therapy. Urinary oligosaccharides were extracted using solid phase extraction (SPE), labeled by fluorescent tag 2-aminobenzamide, and quantified by high-performance liquid chromatography (HPLC) with fluorescence detector (FLD). The identity of peaks was determined by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition, the levels of urinary mannose-rich oligosaccharides were also quantified by 1H nuclear magnetic resonance (NMR) spectroscopy. The data were analyzed using one-tailed paired t-test and Pearson's correlation tests. RESULTS: Compared to levels before the administration of therapy, an approximately two-folds decrease in total mannose-rich oligosaccharides after one month of treatment was observed by NMR and HPLC. After four months, an approximately ten-folds significant decrease in total urinary mannose-rich oligosaccharides was detected, suggesting therapy effectiveness. A significant decrease in the levels of oligosaccharides with 7-9 mannose units was detected by HPLC. CONCLUSIONS: The application of both HPLC-FLD and NMR in quantification of oligosaccharide biomarkers is a suitable approach for monitoring of therapy efficacy in alpha-mannosidosis patients.

N-Glycoprofiling of SLC35A2-CDG: Patient with a Novel Hemizygous Variant.

Congenital disorders of glycosylation (CDG) are a group of rare inherited metabolic disorders caused by a defect in the process of protein glycosylation. In this work, we present a comprehensive glycoprofile analysis of a male patient with a novel missense variant in the SLC35A2 gene, coding a galactose transporter that translocates UDP-galactose from the cytosol to the lumen of the endoplasmic reticulum and Golgi apparatus. Isoelectric focusing of serum transferrin, which resulted in a CDG type II pattern, was followed by structural analysis of transferrin and serum N-glycans, as well as the analysis of apolipoprotein CIII O-glycans by mass spectrometry. An abnormal serum N-glycoprofile with significantly increased levels of agalactosylated (Hex3HexNAc4-5 and Hex3HexNAc5Fuc1) and monogalactosylated (Hex4HexNAc4 ± NeuAc1) N-glycans was observed. Additionally, whole exome sequencing and Sanger sequencing revealed de novo hemizygous c.461T > C (p.Leu154Pro) mutation in the SLC35A2 gene. Based on the combination of biochemical, analytical, and genomic approaches, the set of distinctive N-glycan biomarkers was characterized. Potentially, the set of identified aberrant N-glycans can be specific for other variants causing SLC35A2-CDG and can distinguish this disorder from the other CDGs or other defects in the galactose metabolism.

Application of MALDI-TOF Mass Spectrometry for Non-invasive Diagnostics of Mucopolysaccharidosis IIIA

Abstract Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) caused by deficiency of lysosomal N-sulphoglucosamine sulphohydrolase, which is one of four enzymes involved in heparan sulfate degradation. Traditional methods used for MPS IIIA diagnostics usually constitute of selective screening, based on the analysis of urinary glycosaminoglycans, further enzymatic assays in leukocytes, and mutation analysis. Nowadays, some LSDs, including mucopolysaccharidoses, can be precisely diagnosed by mass spectrometry-based techniques. Up to this date, there are no comprehensive studies of MPS IIIA diagnostics by MALDI-TOF analysis of free oligosaccharides in urine published. In the presented work, MALDI-TOF/TOF analysis of permethylated oligosaccharides was performed to obtain the set of glyco-biomarkers that together form the specific fingerprint of this disease. Early and accurate diagnostics of MPS IIIA is crucial to stabilize the progressive cellular damage and improve the overall well-being of patients.

Defining the Functional Interactome of Spliceosome-Associated G-Patch Protein Gpl1 in the Fission Yeast Schizosaccharomyces pombe.

Pre-mRNA splicing plays a fundamental role in securing protein diversity by generating multiple transcript isoforms from a single gene. Recently, it has been shown that specific G-patch domain-containing proteins are critical cofactors involved in the regulation of splicing processes. In this study, using the knock-out strategy, affinity purification and the yeast-two-hybrid assay, we demonstrated that the spliceosome-associated G-patch protein Gpl1 of the fission yeast S. pombe mediates interactions between putative RNA helicase Gih35 (SPAC20H4.09) and WD repeat protein Wdr83, and ensures their binding to the spliceosome. Furthermore, RT-qPCR analysis of the splicing efficiency of deletion mutants indicated that the absence of any of the components of the Gpl1-Gih35-Wdr83 complex leads to defective splicing of fet5 and pwi1, the reference genes whose unspliced isoforms harboring premature stop codons are targeted for degradation by the nonsense-mediated decay (NMD) pathway. Together, our results shed more light on the functional interactome of G-patch protein Gpl1 and revealed that the Gpl1-Gih35-Wdr83 complex plays an important role in the regulation of pre-mRNA splicing in S. pombe.

Blockade of TMPRSS2-mediated priming of SARS-CoV-2 by lactoferricin.

In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The transmembrane protease serine 2 (TMPRSS2), that is responsible for proteolytic priming of the SARS-CoV-2 spike protein, appears as a rational therapeutic target. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Previously, we identified the human milk glycoprotein lactoferrin as a natural inhibitor of plasminogen conversion to plasmin, a serine protease homologous to TMPRSS2. Here, we tested whether lactoferrin and lactoferricin, a biologically active natural peptide produced by pepsin-mediated digestion of lactoferrin, together with synthetic peptides derived from lactoferrin, were able to block TMPRSS2 and SARS-CoV-2 infection. Particularly, we revealed that both lactoferricin and the N-terminal synthetic peptide pLF1 significantly inhibited: i) proteolytic activity of TMPRSS2 and plasmin, ii) proteolytic processing of the SARS-CoV-2 spike protein, and iii) SARS-CoV-2 infection of SARS-CoV-2-permissive cells. Thus, natural and synthetic peptides derived from lactoferrin represent feasible candidates for supporting prevention and treatment of COVID-19.

Fragmentation analysis of O-specific polysaccharide from bacteria Vibrio cholerae O139 by MALDI-TOF and LC/ESI-MS/MS.

Cholera is a life-threatening diarrhoeal disease caused by ingestion of Vibrio cholerae. There are at least 200 serogroups of V. cholerae but only two of them are causing epidemics - O1 and O139 serogroups. Fragmentation analysis of O-antigen, also known as O-specific polysaccharide (OSP), from lipopolysaccharide (LPS) is important to obtain new information about its structure, such as fragmentation patterns and fragment structures. In the present study, OSP and core (OSPc) structure from V. cholerae O139 was studied using matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) and direct injection electrospray ionization (ESI)-MS methods. MALDI-TOF analysis was performed in positive-ion reflectron mode, while ESI-MS was performed in negative ionization mode. ESI-MS analysis was followed by ESI-MS/MS analysis. Using this analytical approach, we managed to obtain two possible fragmentation pathways of OSP from V. cholerae O139. Mutual sign of these two pathways is shortening the length of the oligosaccharide by neutral loss of monosaccharide residues. Additionally, liquid chromatography-MS analysis was performed to separate depicted molecular forms of OSPc.

Deep Insights into the Specific Evolution of Fungal Hybrid B Heme Peroxidases.

In this study, we focus on a detailed bioinformatics analysis of hyBpox genes, mainly within the genomes of Sclerotiniaceae (Ascomycota, Leotiomycetes), which is a specifically evolved fungal family of necrotrophic host generalists and saprophytic or biotrophic host specialists. Members of the genus Sclerotium produce only sclerotia and no fruiting bodies or spores. Thus, their physiological role for peroxidases remains open. A representative species, S. cepivorum, is a dangerous plant pathogen causing white rot in Allium species, particularly in onions, leeks, and garlic. On a worldwide basis, the white rot caused by this soil-borne fungus is apparently the most serious threat to Allium-crop production. We have also found very similar peroxidase sequences in the related fungus S. sclerotiorum, although with minor yet important modifications in the architecture of its active centre. The presence of ScephyBpox1-specific mRNA was confirmed by transcriptomic analysis. The presence of Hybrid B peroxidase at the protein level as the sole extracellular peroxidase of this fungus was confirmed in the secretome of S. cepivorum through detailed proteomic analyses. This prompted us to systematically search for all available genes coding for Hybrid B heme peroxidases in the whole fungal family of Sclerotiniaceae. We present here a reconstruction of their molecular phylogeny and analyse the unique aspects of their conserved-sequence features and structural folds in corresponding ancestral sequences.

Transcriptome and proteome profiling reveals complex adaptations of Candida parapsilosis cells assimilating hydroxyaromatic carbon sources.

Many fungal species utilize hydroxyderivatives of benzene and benzoic acid as carbon sources. The yeast Candida parapsilosis metabolizes these compounds via the 3-oxoadipate and gentisate pathways, whose components are encoded by two metabolic gene clusters. In this study, we determine the chromosome level assembly of the C. parapsilosis strain CLIB214 and use it for transcriptomic and proteomic investigation of cells cultivated on hydroxyaromatic substrates. We demonstrate that the genes coding for enzymes and plasma membrane transporters involved in the 3-oxoadipate and gentisate pathways are highly upregulated and their expression is controlled in a substrate-specific manner. However, regulatory proteins involved in this process are not known. Using the knockout mutants, we show that putative transcriptional factors encoded by the genes OTF1 and GTF1 located within these gene clusters function as transcriptional activators of the 3-oxoadipate and gentisate pathway, respectively. We also show that the activation of both pathways is accompanied by upregulation of genes for the enzymes involved in ß-oxidation of fatty acids, glyoxylate cycle, amino acid metabolism, and peroxisome biogenesis. Transcriptome and proteome profiles of the cells grown on 4-hydroxybenzoate and 3-hydroxybenzoate, which are metabolized via the 3-oxoadipate and gentisate pathway, respectively, reflect their different connection to central metabolism. Yet we find that the expression profiles differ also in the cells assimilating 4-hydroxybenzoate and hydroquinone, which are both metabolized in the same pathway. This finding is consistent with the phenotype of the Otf1p-lacking mutant, which exhibits impaired growth on hydroxybenzoates, but still utilizes hydroxybenzenes, thus indicating that additional, yet unidentified transcription factor could be involved in the 3-oxoadipate pathway regulation. Moreover, we propose that bicarbonate ions resulting from decarboxylation of hydroxybenzoates also contribute to differences in the cell responses to hydroxybenzoates and hydroxybenzenes. Finally, our phylogenetic analysis highlights evolutionary paths leading to metabolic adaptations of yeast cells assimilating hydroxyaromatic substrates.

Tandem affinity purification protocol for isolation of protein complexes from Schizosaccharomyces pombe.

Many cellular processes require the activities of complex molecular machines composed of several protein subunits. Insights into these systems can be gained by isolation of protein complexes followed by in vitro analyses determining the identity, posttranslational modifications, and interactions among proteins. Here, we present a protocol for tandem affinity purification (TAP) of protein complexes from the fission yeast Schizosaccharomyces pombe. The protocol employs cells expressing C-terminally TAP-tagged proteins and is suitable for the analysis of purified proteins by mass spectrometry. For complete information on the use and execution of this protocol, please refer to Cipakova et al. (2019).

Highly flexible metabolism of the marine euglenozoan protist Diplonema papillatum.

BACKGROUND: The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. RESULTS: We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, ß-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. CONCLUSIONS: The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids.

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.