Senescence of endothelial cells promotes phenotypic changes in adventitial fibroblasts: possible implications for vascular aging.

Aging is the most important risk factor for the development of cardiovascular diseases. Senescent cells release plethora of factors commonly known as the senescence-associated secretory phenotype, which can modulate the normal function of the vascular wall. It is currently not well understood if and how endothelial cell senescence can affect adventitial niche. The aim of this study was to characterize oxidative stress-induced endothelial cells senescence and identify their paracrine effects on the primary cell type of the adventitia, the fibroblasts. Human aortic endothelial cells (HAEC) were treated with hydrogen peroxide to induce premature senescence. Mass spectrometry analysis identified several proteomic changes in senescent HAEC with top upregulated secretory protein growth differentiation factor 15 (GDF-15). Treatment of the human adventitial fibroblast cell line (hAdv cells) with conditioned medium (CM) from senescent HAEC resulted in alterations in the proteome of hAdv cells identified in mass spectrometry analysis. Majority of differentially expressed proteins in hAdv cells treated with CM from senescent HAEC were involved in the uptake and metabolism of lipoproteins, mitophagy and ferroptosis. We next analyzed if some of these changes and pathways might be regulated by GDF-15. We found that recombinant GDF-15 affected some ferroptosis-related factors (e.g. ferritin) and decreased oxidative stress in the analyzed adventitial fibroblast cell line, but it had no effect on erastin-induced cell death. Contrary, silencing of GDF-15 in hAdv cells was protective against this ferroptotic stimuli. Our findings can be of importance for potential therapeutic strategies targeting cell senescence or ferroptosis to alleviate vascular diseases.

Prognostic role of the updated Kiel classification in canine high-grade T-cell lymphomas.

BACKGROUND: The aim of this study was to determine and describe the prognostic role of the morphological subtype determined according to the updated Kiel classification in dogs with high-grade T-cell lymphomas (HGTCLs) depending on the treatment applied. OBJECTIVES: The HGTCLs were classified into three subtypes according to the updated Kiel classification: pleomorphic mixed (PM), lymphoblastic lymphoma/acute lymphoblastic leukaemia and plasmacytoid (P). The treatment was divided into a palliative therapy (PlT) group and a chemotherapy (ChT) group. METHODS: The study was conducted between 2009 and 2017, and it enrolled 58 dogs in which cytomorphological and immunocytochemistry diagnoses were HGTCL. RESULTS: Overall survival (OS) was significantly longer in the ChT group (median OS-4 months, interquartile range [IQR] from 2 to 8 months) than in the PlT group (median OS-6 weeks, IQR from 1 week to 3 months). In the PlT group, PM subtype and glucocorticosteroids (GCSs) treatment proved significantly and independently linked to longer OS and approximately three-fold lower risk of death during the study period (adjusted hazard ratio [HRadj] = 0.26, confidence interval [CI] 95%: 0.08-0.81; p = 0.020 and HRadj = 0.30, CI 95%: 0.11-0.77; p = 0.013, respectively), although due to small group size, precision of estimations was poor (wide CI 95%). In the ChT group, >7 days elapsing between diagnosis and the beginning of chemotherapy and GCS treatment prior to chemotherapy were significantly associated with lower chance of complete remission (CR; p = 0.034 for both); GCS treatment prior to chemotherapy was significantly associated with shorter OS (p = 0.016); chemotherapy based on the modified CHOP protocol was significantly associated with higher chance of CR (p = 0.034) and longer OS (p = 0.039); and CR was significantly linked to longer OS (p = 0.001). CLINICAL SIGNIFICANCE: The morphological subtype of HGTCL has some prognostic value in dogs treated palliatively (with PM subtype associated with longer OS than P subtype); however, this effect is no longer visible when a dog is treated with chemotherapy.

Cryo-EM structures of the human Elongator complex at work.

tRNA modifications affect ribosomal elongation speed and co-translational folding dynamics. The Elongator complex is responsible for introducing 5-carboxymethyl at wobble uridine bases (cm5U34) in eukaryotic tRNAs. However, the structure and function of human Elongator remain poorly understood. In this study, we present a series of cryo-EM structures of human ELP123 in complex with tRNA and cofactors at four different stages of the reaction. The structures at resolutions of up to 2.9 Å together with complementary functional analyses reveal the molecular mechanism of the modification reaction. Our results show that tRNA binding exposes a universally conserved uridine at position 33 (U33), which triggers acetyl-CoA hydrolysis. We identify a series of conserved residues that are crucial for the radical-based acetylation of U34 and profile the molecular effects of patient-derived mutations. Together, we provide the high-resolution view of human Elongator and reveal its detailed mechanism of action.

Phototropin2 3'UTR overlaps with the AT5G58150 gene encoding an inactive RLK kinase.

BACKGROUND: This study examines the biological implications of an overlap between two sequences in the Arabidopsis genome, the 3'UTR of the PHOT2 gene and a putative AT5G58150 gene, encoded on the complementary strand. AT5G58150 is a probably inactive protein kinase that belongs to the transmembrane, leucine-rich repeat receptor-like kinase family. Phot2 is a membrane-bound UV/blue light photoreceptor kinase. Thus, both proteins share their cellular localization, on top of the proximity of their loci. RESULTS: The extent of the overlap between 3'UTR regions of AT5G58150 and PHOT2 was found to be 66 bp, using RACE PCR. Both the at5g58150 T-DNA SALK_093781C (with insertion in the promoter region) and 35S::AT5G58150-GFP lines overexpress the AT5G58150 gene. A detailed analysis did not reveal any substantial impact of PHOT2 or AT5G58150 on their mutual expression levels in different light and osmotic stress conditions. AT5G58150 is a plasma membrane protein, with no apparent kinase activity, as tested on several potential substrates. It appears not to form homodimers and it does not interact with PHOT2. Lines that overexpress AT5G58150 exhibit a greater reduction in lateral root density due to salt and osmotic stress than wild-type plants, which suggests that AT5G58150 may participate in root elongation and formation of lateral roots. In line with this, mass spectrometry analysis identified proteins with ATPase activity, which are involved in proton transport and cell elongation, as putative interactors of AT5G58150. Membrane kinases, including other members of the LRR RLK family and BSK kinases (positive regulators of brassinosteroid signalling), can also act as partners for AT5G58150. CONCLUSIONS: AT5G58150 is a membrane protein that does not exhibit measurable kinase activity, but is involved in signalling through interactions with other proteins. Based on the interactome and root architecture analysis, AT5G58150 may be involved in plant response to salt and osmotic stress and the formation of roots in Arabidopsis.

Similarities and Differences in the Protein Composition of Cutaneous Melanoma Cells and Their Exosomes Identified by Mass Spectrometry.

Intercellular transport of proteins mediated by extracellular vesicles (EVs)-exosomes and ectosomes-is one of the factors facilitating carcinogenesis. Therefore, the research on protein cargo of melanoma-derived EVs may provide a better understanding of the mechanisms involved in melanoma progression and contribute to the development of alternative biomarkers. Proteomic data on melanoma-derived EVs are very limited. The shotgun nanoLC-MS/MS approach was applied to analyze the protein composition of primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) cutaneous melanoma cells and exosomes released by them. All cells secreted homogeneous populations of exosomes that shared a characteristic set of proteins. In total, 3514 and 1234 unique proteins were identified in melanoma cells and exosomes, respectively. Gene ontology analysis showed enrichment in several cancer-related categories, including cell proliferation, migration, negative regulation of apoptosis, and angiogenesis. The obtained results broaden our knowledge on the role of selected proteins in exosome biology, as well as their functional role in the development and progression of cutaneous melanoma. The results may also inspire future studies on the clinical potential of exosomes.

Coordinated reprogramming of renal cancer transcriptome, metabolome and secretome associates with immune tumor infiltration.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cancer. The molecules (proteins, metabolites) secreted by tumors affect their extracellular milieu to support cancer progression. If secreted in amounts detectable in plasma, these molecules can also serve as useful, minimal invasive biomarkers. The knowledge of ccRCC tumor microenvironment is fragmentary. In particular, the links between ccRCC transcriptome and the composition of extracellular milieu are weakly understood. In this study, we hypothesized that ccRCC transcriptome is reprogrammed to support alterations in tumor microenvironment. Therefore, we comprehensively analyzed ccRCC extracellular proteomes and metabolomes as well as transcriptomes of ccRCC cells to find molecules contributing to renal tumor microenvironment. METHODS: Proteomic and metabolomics analysis of conditioned media isolated from normal kidney cells as well as five ccRCC cell lines was performed using mass spectrometry, with the following ELISA validation. Transcriptomic analysis was done using microarray analysis and validated using real-time PCR. Independent transcriptomic and proteomic datasets of ccRCC tumors were used for the analysis of gene and protein expression as well as the level of the immune infiltration. RESULTS: Renal cancer secretome contained 85 proteins detectable in human plasma, consistently altered in all five tested ccRCC cell lines. The top upregulated extracellular proteins included SPARC, STC2, SERPINE1, TGFBI, while downregulated included transferrin and DPP7. The most affected extracellular metabolites were increased 4-hydroxy-proline, succinic acid, cysteine, lactic acid and downregulated glutamine. These changes were associated with altered expression of genes encoding the secreted proteins (SPARC, SERPINE1, STC2, DPP7), membrane transporters (SLC16A4, SLC6A20, ABCA12), and genes involved in protein trafficking and secretion (KIF20A, ANXA3, MIA2, PCSK5, SLC9A3R1, SYTL3, and WNTA7). Analogous expression changes were found in ccRCC tumors. The expression of SPARC predicted the infiltration of ccRCC tumors with endothelial cells. Analysis of the expression of the 85 secretome genes in > 12,000 tumors revealed that SPARC is a PanCancer indicator of cancer-associated fibroblasts' infiltration. CONCLUSIONS: Transcriptomic reprogramming of ccRCC supports the changes in an extracellular milieu which are associated with immune infiltration. The proteins identified in our study represent valuable cancer biomarkers detectable in plasma.

E2/E3-independent ubiquitin-like protein conjugation by Urm1 is directly coupled to cysteine persulfidation.

Post-translational modifications by ubiquitin-like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin-related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin-like protein activator 4). While Urm1 has also been observed to conjugate to target proteins like other UBLs, the molecular mechanism of its attachment remains unknown. Here, we reconstitute the covalent attachment of thiocarboxylated Urm1 to various cellular target proteins in vitro, revealing that, unlike other known UBLs, this process is E2/E3-independent and requires oxidative stress. Furthermore, we present the crystal structures of the peroxiredoxin Ahp1 before and after the covalent attachment of Urm1. Surprisingly, we show that urmylation is accompanied by the transfer of sulfur to cysteine residues in the target proteins, also known as cysteine persulfidation. Our results illustrate the role of the Uba4-Urm1 system as a key evolutionary link between prokaryotic SCPs and the UBL modifications observed in modern eukaryotes.

Comparative Proteomic Profiling of Ectosomes Derived from Thyroid Carcinoma and Normal Thyroid Cells Uncovers Multiple Proteins with Functional Implications in Cancer.

Proteins carried by tumor-derived ectosomes play an important role in cancer progression, and are considered promising diagnostic markers. In the present study, a shotgun nanoLC-MS/MS proteomic approach was applied to profile and compare the protein content of ectosomes released in vitro by normal human thyroid follicular epithelial Nthy-ori 3-1 cells and human anaplastic thyroid carcinoma (TC) 8305C cells. Additionally, the pro-migratory and pro-proliferative effects of Nthy-ori 3-1- and 8305C-derived ectosomes exerted on the recipient cells were assessed in wound closure and Alamar Blue assays. A total of 919 proteins were identified in all replicates of 8305C-derived ectosomes, while Nthy-ori 3-1-derived ectosomes contained a significantly lower number of 420 identified proteins. Qualitative analysis revealed 568 proteins present uniquely in 8305C-derived ectosomes, suggesting their applicability in TC diagnosis and management. In addition, 8305C-derived ectosomes were able to increase the proliferation and motility rates of the recipient cells, likely due to the ectosomal transfer of the identified cancer-promoting molecules. Our description of ectosome protein content and its related functions provides the first insight into the role of ectosomes in TC development and progression. The results also indicate the applicability of some of these ectosomal proteins for further investigation regarding their potential as circulating TC biomarkers.

Functional Protein Composition in Femoral Glands of Sand Lizards (Lacerta agilis).

Proteins are ubiquitous macromolecules that display a vast repertoire of chemical and enzymatic functions, making them suitable candidates for chemosignals, used in intraspecific communication. Proteins are present in the skin gland secretions of vertebrates but their identity, and especially, their functions, remain largely unknown. Many lizard species possess femoral glands, i.e., epidermal organs primarily involved in the production and secretion of chemosignals, playing a pivotal role in mate choice and intrasexual communication. The lipophilic fraction of femoral glands has been well studied in lizards. In contrast, proteins have been the focus of only a handful of investigations. Here, we identify and describe inter-individual expression patterns and the functionality of proteins present in femoral glands of male sand lizards (Lacerta agilis) by applying mass spectrometry-based proteomics. Our results show that the total number of proteins varied substantially among individuals. None of the identified femoral gland proteins could be directly linked to chemical communication in lizards, although this result hinges on protein annotation in databases in which squamate semiochemicals are poorly represented. In contrast to our expectations, the proteins consistently expressed across individuals were related to the immune system, antioxidant activity and lipid metabolism as their main functions, showing that proteins in reptilian epidermal glands may have other functions besides chemical communication. Interestingly, we found expression of the Major Histocompatibility Complex (MHC) among the multiple and diverse biological processes enriched in FGs, tentatively supporting a previous hypothesis that MHC was coopted for semiochemical function in sand lizards, specifically in mate recognition. Our study shows that mass spectrometry-based proteomics are a powerful tool for characterizing and deciphering the role of proteins secreted by skin glands in non-model vertebrates.

Hyperosmolarity adversely impacts recombinant protein synthesis by Yarrowia lipolytica-molecular background revealed by quantitative proteomics.

In this research, we were interested in answering a question whether subjecting a Yarrowia lipolytica strain overproducing a recombinant secretory protein (rs-Prot) to pre-optimized stress factors may enhance synthesis of the rs-Prot. Increased osmolarity (3 Osm kg-1) was the primary stress factor implemented alone or in combination with decreased temperature (20 °C), known to promote synthesis of rs-Prots. The treatments were executed in batch bioreactor cultures, and the cellular response was studied in terms of culture progression, gene expression and global proteomics, to get insight into molecular bases underlying an awaken reaction. Primarily, we observed that hyperosmolarity executed by high sorbitol concentration does not enhance synthesis of the rs-Prot but increases its transcription. Expectedly, hyperosmolarity induced synthesis of polyols at the expense of citric acid synthesis and growth, which was severely limited. A number of stress-related proteins were upregulated, including heat-shock proteins (HSPs) and aldo-keto reductases, as observed at transcriptomics and proteomics levels. Concerted downregulation of central carbon metabolism, including glycolysis, tricarboxylic acid cycle and fatty acid synthesis, highlighted redirection of carbon fluxes. Elevated abundance of HSPs and osmolytes did not outbalance the severe limitation of protein synthesis, marked by orchestrated downregulation of translation (elongation factors, several aa-tRNA synthetases), amino acid biosynthesis and ribosome biogenesis in response to the hyperosmolarity. Altogether we settled that increased osmolarity is not beneficial for rs-Prots synthesis in Y. lipolytica, even though some elements of the response could assist this process. Insight into global changes in the yeast proteome under the treatments is provided. KEY POINTS: • Temp enhances, but Osm decreases rs-Prots synthesis by Y. lipolytica. • Enhanced abundance of HSPs and osmolytes is overweighted by limited translation. • Global proteome under Osm, Temp and Osm Temp treatments was studied.

Changes in the Cell Wall Proteome of Leaves in Response to High Temperature Stress in Brachypodium distachyon.

High temperature stress leads to complex changes to plant functionality, which affects, i.a., the cell wall structure and the cell wall protein composition. In this study, the qualitative and quantitative changes in the cell wall proteome of Brachypodium distachyon leaves in response to high (40 °C) temperature stress were characterised. Using a proteomic analysis, 1533 non-redundant proteins were identified from which 338 cell wall proteins were distinguished. At a high temperature, we identified 46 differentially abundant proteins, and of these, 4 were over-accumulated and 42 were under-accumulated. The most significant changes were observed in the proteins acting on the cell wall polysaccharides, specifically, 2 over- and 12 under-accumulated proteins. Based on the qualitative analysis, one cell wall protein was identified that was uniquely present at 40 °C but was absent in the control and 24 proteins that were present in the control but were absent at 40 °C. Overall, the changes in the cell wall proteome at 40 °C suggest a lower protease activity, lignification and an expansion of the cell wall. These results offer a new insight into the changes in the cell wall proteome in response to high temperature.

Proteomic Profiling of Ectosomes Derived from Paired Urothelial Bladder Cancer and Normal Cells Reveals the Presence of Biologically-Relevant Molecules.

Protein content of extracellular vesicles (EVs) can modulate different processes during carcinogenesis. Novel proteomic strategies have been applied several times to profile proteins present in exosomes released by urothelial bladder cancer (UBC) cells. However, similar studies have not been conducted so far on another population of EVs, i.e., ectosomes. In the present study we used a shotgun nanoLC-MS/MS proteomic approach to investigate the protein content of ectosomes released in vitro by T-24 UBC cells and HCV-29 normal ureter epithelial cells. In addition, cancer-promoting effects exerted by UBC-derived ectosomes on non-invasive cells in terms of cell proliferation and migratory properties were assessed. In total, 1158 proteins were identified in T-24-derived ectosomes, while HCV-29-derived ectosomes contained a lower number of 259 identified proteins. Qualitative analysis revealed 938 proteins present uniquely in T-24-derived ectosomes, suggesting their potential applications in bladder cancer management as diagnostic and prognostic biomarkers. In addition, T-24-derived ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of the identified cancer-promoting molecules. The present study provided a focused identification of biologically relevant proteins in UBC-derived ectosomes, confirming their role in UBC development and progression, and their applicability for further biomarker-oriented studies in preclinical or clinical settings.

Proteome Analysis of PC12 Cells Reveals Alterations in Translation Regulation and Actin Signaling Induced by Clozapine.

Although antipsychotics are routinely used in the treatment of schizophrenia for the last decades, their precise mechanism of action is still unclear. In this study, we investigated changes in the PC12 cells' proteome under the influence of clozapine, risperidone, and haloperidol to identify protein pathways regulated by antipsychotics. Analysis of the protein profiles in two time points: after 12 and 24 h of incubation with drugs revealed significant alterations in 510 proteins. Further canonical pathway analysis revealed an inhibition of ciliary trophic factor signaling after treatment with haloperidol and showed a decrease in acute phase response signaling in the risperidone group. Interestingly, all tested drugs have caused changes in PC12 proteome which correspond to inhibition of cytokines: tumor necrosis factor (TNF) and transforming growth factor beta 1 (TGF-ß1). We also found that the 12-h incubation with clozapine caused up-regulation of protein kinase A signaling and translation machinery. After 24 h of treatment with clozapine, the inhibition of the actin cytoskeleton signaling and Rho proteins signaling was revealed. The obtained results suggest that the mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) play a central role in the signal transduction of clozapine.

Perturbations of the hepatic proteome behind the onset of metabolic disorders in mouse offspring developed following embryo manipulation.

Assisted Reproductive Technologies (ART) allowed the births of >8 million babies worldwide. Even if ART children are healthy at birth, several studies reported that ART may cause changes in foetal programming, leading to an increased predisposition to metabolic disorders in adulthood. Previous studies on mouse model showed obesity, glucose intolerance, and hepatic lipid accumulation in ART offspring. A cumulative effect of the different components of ART protocol has been previously described, for example, in the occurrence of epigenetic defects. Here, we investigated whether there is a cumulative effect of embryo transfer (ET), in vitro culture (IVC) and blastomere biopsy (BB) in the onset of metabolic disorders in mouse offspring vs those naturally conceived (Control - CTR). To this aim, proteomic analysis was performed on the livers from adult mouse offspring developed following ET, IVC and BB vs CTR. We observed deregulated expression of proteins involved in lipid, carbohydrate, energy metabolisms and cellular processes in ART offspring. Moreover, we found increased body weight in all ART offspring while i) insulin resistance in BB male, ii) females glucose intolerance and high level of triglycerides and cholesterol in BB females and iii) low levels of interleukin-6 in BB, IVC and ET males. In conclusion, our study suggests that the use of various embryo manipulations influences the metabolic health of adult offspring, resulting in an increased predisposition to hepatic diseases and metabolic syndrome in a sex-specific manner.

Structural Determinants of Substrate Specificity of SplF Protease from Staphylococcus aureus.

Accumulating evidence suggests that six proteases encoded in the spl operon of a dangerous human pathogen, Staphylococcus aureus, may play a role in virulence. Interestingly, SplA, B, D, and E have complementary substrate specificities while SplF remains to be characterized in this regard. Here, we describe the prerequisites of a heterologous expression system for active SplF protease and characterize the enzyme in terms of substrate specificity and its structural determinants. Substrate specificity of SplF is comprehensively profiled using combinatorial libraries of peptide substrates demonstrating strict preference for long aliphatic sidechains at the P1 subsite and significant selectivity for aromatic residues at P3. The crystal structure of SplF was provided at 1.7 Å resolution to define the structural basis of substrate specificity of SplF. The obtained results were compared and contrasted with the characteristics of other Spl proteases determined to date to conclude that the spl operon encodes a unique extracellular proteolytic system.

Same but different - Molecular comparison of human KTI12 and PSTK.

Kti12 and PSTK are closely related and highly similar proteins implicated in different aspects of tRNA metabolism. Kti12 has been identified as an essential regulatory factor of the Elongator complex, involved in the modification of uridine bases in eukaryotic tRNAs. PSTK phosphorylates the tRNASec-bound amino acid serine, which is required to synthesize selenocysteine. Kti12 and PSTK have previously been studied independently in various organisms, but only appear simultaneously in some animalia, including humans. As Kti12- and PSTK-related pathways are clinically relevant, it is of prime importance to understand their biological functions and mutual relationship in humans. Here, we use different tRNA substrates to directly compare the enzymatic activities of purified human KTI12 and human PSTK proteins. Our complementary Co-IP and BioID2 approaches in human cells confirm that Elongator is the main interaction partner of KTI12 but additionally indicate potential links to proteins involved in vesicular transport, RNA metabolism and deubiquitination. Moreover, we identify and validate a yet uncharacterized interaction between PSTK and γ-taxilin. Foremost, we demonstrate that human KTI12 and PSTK do not share interactors or influence their respective biological functions. Our data provide a comprehensive analysis of the regulatory networks controlling the activity of the human Elongator complex and selenocysteine biosynthesis.

Destabilisation of the structure of transthyretin is driven by Ca2.

Tetrameric transthyretin (TTR) transports thyroid hormones and retinol in plasma and cerebrospinal fluid and performs protective functions under stress conditions. Ageing and mutations result in TTR destabilisation and the formation of the amyloid deposits that dysregulate Ca2+ homeostasis. Our aim was to determine whether Ca2+ affects the structural stability of TTR. We show, using multiple techniques, that Ca2+ does not induce prevalent TTR dissociation and/or oligomerisation. However, in the presence of Ca2+, TTR exhibits altered conformational flexibility and different interactions with the solvent molecules. These structural changes lead to the formation of the sub-populations of non-native TTR conformers and to the destabilisation of the structure of TTR. Moreover, the sub-population of TTR molecules undergoes fragmentation that is augmented by Ca2+. We postulate that Ca2+ constitutes the structural and functional switch between the native and non-native forms of TTR, and therefore tip the balance towards age-dependent pathological calcification.

Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.

The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.

SNAIL Promotes Metastatic Behavior of Rhabdomyosarcoma by Increasing EZRIN and AKT Expression and Regulating MicroRNA Networks.

Rhabdomyosarcoma (RMS) is a predominant soft tissue tumor in children and adolescents. For high-grade RMS with metastatic involvement, the 3-year overall survival rate is only 25 to 30%. Thus, understanding the regulatory mechanisms involved in promoting the metastasis of RMS is important. Here, we demonstrate for the first time that the SNAIL transcription factor regulates the metastatic behavior of RMS both in vitro and in vivo. SNAIL upregulates the protein expression of EZRIN and AKT, known to promote metastatic behavior, by direct interaction with their promoters. Our data suggest that SNAIL promotes RMS cell motility, invasion and chemotaxis towards the prometastatic factors: HGF and SDF-1 by regulating RHO, AKT and GSK3b activity. In addition, miRNA transcriptome analysis revealed that SNAIL-miRNA axis regulates processes associated with actin cytoskeleton reorganization. Our data show a novel role of SNAIL in regulating RMS cell metastasis that may also be important in other mesenchymal tumor types and clearly suggests SNAIL as a promising new target for future RMS therapies.

Analysis of a Nuclear Intrinsically Disordered Proteome.

Intrinsically disordered proteins (IDPs) play crucial roles in cell functioning, although they do not possess defined three-dimensional architecture. They are highly abundant in the cell nucleus, and the vast majority of transcription factors (TFs) contain extended regions of intrinsic disorder. IDPs do not respond to denaturing conditions in a standard manner, and this can be used for their separation from structured proteins. Here we describe a protocol for the isolation and characterization of nuclear IDPs in which heat treatment is used for enrichment of IDPs in samples. The whole workflow comprises the following steps: nuclei isolation from HEK293 (human embryonic kidney) cells, protein extraction, enrichment of IDPs, sample preparation for mass spectrometric analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, in silico assessment of protein disorder, and Gene Ontology analysis.