A heterotypic assembly mechanism regulates CHIP E3 ligase activity.

CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.

In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2'-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2'-O-methylated adenosine, it can be additionally modified to N6,2'-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between 'self' and 'non-self' in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for RNA capping during in vitro transcription. Using this tool, we found that 2'-O-methylation of the second transcribed nucleotide within the mRNA 5' cap influences protein production levels in a cell-specific manner. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2'-O-methylation of the second transcribed nucleotide and the presence of m6Am as the first transcribed nucleotide serve as determinants that define transcripts as 'self' and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation.

Allogenic Adipose-Derived Stem Cells in Diabetic Foot Ulcer Treatment: Clinical Effectiveness, Safety, Survival in the Wound Site, and Proteomic Impact.

Although encouraging results of adipose-derived stem cell (ADSC) use in wound healing are available, the mechanism of action has been studied mainly in vitro and in animals. This work aimed to examine the safety and efficacy of allogenic ADSCs in human diabetic foot ulcer treatment, in combination with the analyses of the wound. Equal groups of 23 participants each received fibrin gel with ADSCs or fibrin gel alone. The clinical effects were assessed at four time points: days 7, 14, 21 and 49. Material collected during debridement from a subset of each group was analyzed for the presence of ADSC donor DNA and proteomic changes. The reduction in wound size was greater at all subsequent visits, significantly on day 21 and 49, and the time to 50% reduction in the wound size was significantly shorter in patients who received ADSCs. Complete healing was achieved at the end of the study in seven patients treated with ADSCs vs. one treated without ADSCs. One week after ADSC application, 34 proteins significantly differentiated the material from both groups, seven of which, i.e., GAPDH, CAT, ACTN1, KRT1, KRT9, SCL4A1, and TPI, positively correlated with the healing rate. We detected ADSC donor DNA up to 21 days after administration. We confirmed ADSC-related improvement in wound healing that correlated with the molecular background, which provides insights into the role of ADSCs in wound healing-a step toward the development of cell-based therapies.

Mass-spectrometric identification of oxidative modifications in plasma-purified plasminogen: Association with hypofibrinolysis in patients with acute pulmonary embolism.

OBJECTIVES: Mechanisms behind disturbed fibrinolysis in pulmonary embolism (PE) are poorly understood. We hypothesized that oxidative stress-induced changes in plasminogen contribute to impaired fibrinolysis in patients with acute PE. METHODS: Oxidative and other modifications were investigated using mass-spectrometry in plasminogen purified from pooled plasma of 5 acute PE patients on admission and after 3 months of anticoagulant treatment, along with plasma clot lysis time, a measure of global efficiency of fibrinolysis, and a stable oxidative stress marker, plasma 8-isoprostane. RESULTS: Twenty sites of oxidation, 3 sites of carbonylation and 4 sites of S-nitrosylation were identified in plasminogen. The intensity of peptides oxidized at cysteine residues with respect to unmodified peptides decreased after 3 months of anticoagulation (p = 0.018). This was not observed for oxidized methionine residues (p = 0.9). Oxidized tryptophan (n = 4) and proline (n = 2), as well as carbonylation at 3 threonine residues were selectively identified in acute PE episode, not after 3 months. This was accompanied by 12.8% decrease in clot lysis time (p = 0.043). Deamidation occurred at the arginine, previously identified to undergo the cleavage by plasminogen activator. Methylated were two lysine-binding sites important for an interaction of plasminogen with fibrin. Other identified modifications involved: glycation, acetylation, phosphorylation, homocysteinylation, carbamylation and dichlorination (88 modifications at 162 sites). CONCLUSIONS: Data suggest that oxidative stress-induced changes in plasminogen molecules may contribute to less effective global fibrinolysis in patients with acute PE. The comprehensive library of posttranslational modifications in plasminogen molecules was provided, including modifications of sites reported to be involved in important biological functions.

Mitochondrial protein biogenesis in the synapse is supported by local translation.

Synapses are the regions of the neuron that enable the transmission and propagation of action potentials on the cost of high energy consumption and elevated demand for mitochondrial ATP production. The rapid changes in local energetic requirements at dendritic spines imply the role of mitochondria in the maintenance of their homeostasis. Using global proteomic analysis supported with complementary experimental approaches, we show that an essential pool of mitochondrial proteins is locally produced at the synapse indicating that mitochondrial protein biogenesis takes place locally to maintain functional mitochondria in axons and dendrites. Furthermore, we show that stimulation of synaptoneurosomes induces the local synthesis of mitochondrial proteins that are transported to the mitochondria and incorporated into the protein supercomplexes of the respiratory chain. Importantly, in a mouse model of fragile X syndrome, Fmr1 KO mice, a common disease associated with dysregulation of synaptic protein synthesis, we observed altered morphology and respiration rates of synaptic mitochondria. That indicates that the local production of mitochondrial proteins plays an essential role in synaptic functions.

Mechanisms of Resistance to Photodynamic Therapy (PDT) in Vulvar Cancer.

Photodynamic therapy (PDT) is a valuable treatment method for vulvar intraepithelial neoplasia (VIN). It allows for the treatment of a multifocal disease with minimal tissue destruction. 5-Aminolevulinic acid (5-ALA) is the most commonly used prodrug, which is converted in the heme pathway to protoporphyrin IX (PpIX), an actual photosensitizer (PS). Unfortunately, not all patients treated with PDT undergo complete remission. The main cause of their failure is resistance to anticancer therapy. In many cancers, resistance to various anticancer treatments is correlated with increased activity of the DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1). Enhanced activity of drug pumps may also affect the effectiveness of therapy. To investigate whether multidrug resistance mechanisms underlie PDT resistance in VIN, porphyrins were isolated from sensitive and resistant vulvar cancer cells and their culture media. APE1 activity was measured, and survival assay after PDT combined with APE1 inhibitor was performed. Our results revealed that resistant cells accumulated and effluxed less porphyrins than sensitive cells, and in response to PDT, resistant cells increased APE1 activity. Moreover, PDT combined with inhibition of APE1 significantly decreased the survival of PDT-resistant cells. This means that resistance to PDT in vulvar cancer may be the result of alterations in the heme synthesis pathway. Moreover, increased APE1 activity may be essential for the repair of PDT-mediated DNA damage, and inhibition of APE1 activity may increase the efficacy of PDT.

Proteome-Wide Analysis of Protein Lysine N-Homocysteinylation in Saccharomyces cerevisiae.

Protein N-homocysteinylation by a homocysteine (Hcy) metabolite, Hcy-thiolactone, is an emerging post-translational modification (PTM) that occurs in all tested organisms and has been linked to human diseases. The yeast Saccharomyces cerevisiae is widely used as a model eukaryotic organism in biomedical research, including studies of protein PTMs. However, patterns of global protein N-homocysteinylation in yeast are not known. Here, we identified 68 in vivo and 197 in vitro N-homocysteinylation sites at protein lysine residues (N-Hcy-Lys). Some of the N-homocysteinylation sites overlap with other previously identified PTM sites. Protein N-homocysteinylation in vivo, induced by supplementation of yeast cultures with Hcy, which elevates Hcy-thiolactone levels, was accompanied by significant changes in the levels of 70 yeast proteins (38 up-regulated and 32 down-regulated) involved in the ribosomal structure, amino acid biosynthesis, and basic cellular pathways. Our study provides the first global survey of N-homocysteinylation and accompanying changes in the yeast proteome caused by elevated Hcy level. These findings suggest that protein N-homocysteinylation and dysregulation of cellular proteostasis may contribute to the toxicity of Hcy in yeast. Homologous proteins and N-homocysteinylation sites are likely to be involved in Hcy-related pathophysiology in humans and experimental animals. Data are available via ProteomeXchange with identifier PXD020821.

HaDeX: an R package and web-server for analysis of data from hydrogen-deuterium exchange mass spectrometry experiments.

MOTIVATION: Hydrogen-deuterium mass spectrometry (HDX-MS) is a rapidly developing technique for monitoring dynamics and interactions of proteins. The development of new devices has to be followed with new software suites addressing emerging standards in data analysis. RESULTS: We propose HaDeX, a novel tool for processing, analysis and visualization of HDX-MS experiments. HaDeX supports a reproducible analytical process, including data exploration, quality control and generation of publication-quality figures. AVAILABILITY AND IMPLEMENTATION: HaDeX is available primarily as a web-server (http://mslab-ibb.pl/shiny/HaDeX/), but its all functionalities are also accessible as the R package (https://CRAN.R-project.org/package=HaDeX) and standalone software (https://sourceforge.net/projects/HaDeX/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Quantitative proteomics revealed C6orf203/MTRES1 as a factor preventing stress-induced transcription deficiency in human mitochondria.

Maintenance of mitochondrial gene expression is crucial for cellular homeostasis. Stress conditions may lead to a temporary reduction of mitochondrial genome copy number, raising the risk of insufficient expression of mitochondrial encoded genes. Little is known how compensatory mechanisms operate to maintain proper mitochondrial transcripts levels upon disturbed transcription and which proteins are involved in them. Here we performed a quantitative proteomic screen to search for proteins that sustain expression of mtDNA under stress conditions. Analysis of stress-induced changes of the human mitochondrial proteome led to the identification of several proteins with poorly defined functions among which we focused on C6orf203, which we named MTRES1 (Mitochondrial Transcription Rescue Factor 1). We found that the level of MTRES1 is elevated in cells under stress and we show that this upregulation of MTRES1 prevents mitochondrial transcript loss under perturbed mitochondrial gene expression. This protective effect depends on the RNA binding activity of MTRES1. Functional analysis revealed that MTRES1 associates with mitochondrial RNA polymerase POLRMT and acts by increasing mitochondrial transcription, without changing the stability of mitochondrial RNAs. We propose that MTRES1 is an example of a protein that protects the cell from mitochondrial RNA loss during stress.

Butyrylcholinesterase-Protein Interactions in Human Serum.

Measuring various biochemical and cellular components in the blood is a routine procedure in clinical practice. Human serum contains hundreds of diverse proteins secreted from all cells and tissues in healthy and diseased states. Moreover, some serum proteins have specific strong interactions with other blood components, but most interactions are probably weak and transient. One of the serum proteins is butyrylcholinesterase (BChE), an enzyme existing mainly as a glycosylated soluble tetramer that plays an important role in the metabolism of many drugs. Our results suggest that BChE interacts with plasma proteins and forms much larger complexes than predicted from the molecular weight of the BChE tetramer. To investigate and isolate such complexes, we developed a two-step strategy to find specific protein-protein interactions by combining native size-exclusion chromatography (SEC) with affinity chromatography with the resin that specifically binds BChE. Second, to confirm protein complexes' specificity, we fractionated blood serum proteins by density gradient ultracentrifugation followed by co-immunoprecipitation with anti-BChE monoclonal antibodies. The proteins coisolated in complexes with BChE were identified by mass spectroscopy. These binding studies revealed that BChE interacts with a number of proteins in the human serum. Some of these interactions seem to be more stable than transient. BChE copurification with ApoA-I and the density of some fractions containing BChE corresponding to high-density lipoprotein cholesterol (HDL) during ultracentrifugation suggest its interactions with HDL. Moreover, we observed lower BChE plasma activity in individuals with severely reduced HDL levels (≤20 mg/dL). The presented two-step methodology for determination of the BChE interactions can facilitate further analysis of such complexes, especially from the brain tissue, where BChE could be involved in the pathogenesis and progression of AD.

Identification of glycated and acetylated lysine residues in human α2-antiplasmin.

BACKGROUND: The post-translational protein modification via lysine residues can significantly alter its function. α2-antiplasmin, a key inhibitor of fibrinolysis, contains 19 lysine residues. AIM: We sought to identify sites of glycation and acetylation in human α2-antiplasmin and test whether the competition might occur on the lysine residues of α2-antiplasmin. METHODS: We analyzed human α2-antiplasmin (1) untreated; (2) incubated with increasing concentrations of ß-d-glucose (0, 5, 10, 50 mM); (3) incubated with 1.6 mM acetylsalicylic acid (ASA) and (4) incubated with 1.6 mM ASA and 50 mM ß-d-glucose, using the ultraperformance liquid chromatography system coupled to mass spectrometer. RESULTS: Eleven glycation sites and 10 acetylation sites were found in α2-antiplasmin. Incubation with ß-d-glucose was associated with glycation of 4 (K-418, K-427, K-434, K-441) out of 6 lysine residues, known to be important for mediating the interaction with plasmin. Glycation and acetylation overlapped at 9 sites in samples incubated with ß-d-glucose or ASA. Incubation with concomitant ASA and ß-d-glucose was associated with the decreased acetylation at all sites overlapping with glycation sites. At K-182 and K-448, decreased acetylation was associated with increased glycation when compared with α2-antiplasmin incubated with 50 mM ß-d-glucose alone. Although K-24 located in the proximity of the α2-antiplasmin cleavage site, was found to be only acetylated, incubation with ASA and 50 mM ß-d-glucose was associated the absence of acetylation at that site. CONCLUSION: Human α2-antiplasmin is glycated and acetylated at several sites, with the possible competition between acetylation and glycation at K-182 and K-448. Our finding suggests possibly relevant alterations to α2-antiplasmin function at high glycemia and during aspirin use.

Effects of Graphene Oxide Nanofilm and Chicken Embryo Muscle Extract on Muscle Progenitor Cell Differentiation and Contraction.

Finding an effective muscle regeneration technique is a priority for regenerative medicine. It is known that the key factors determining tissue formation include cells, capable of proliferating and/or differentiating, a niche (surface) allowing their colonization and growth factors. The interaction between these factors, especially between the surface of the artificial niche and growth factors, is not entirely clear. Moreover, it seems that the use of a complex of complementary growth factors instead of a few strictly defined ones could increase the effectiveness of tissue maturation, including muscle tissue. In this study, we evaluated whether graphene oxide (GO) nanofilm, chicken embryo muscle extract (CEME), and GO combined with CEME would affect the differentiation and functional maturation of muscle precursor cells, as well as the ability to spontaneously contract a pseudo-tissue muscle. CEME was extracted on day 18 of embryogenesis. Muscle cells obtained from an 8-day-old chicken embryo limb bud were treated with GO and CEME. Cell morphology and differentiation were observed using different microscopy methods. Cytotoxicity and viability of cells were measured by lactate dehydrogenase and Vybrant Cell Proliferation assays. Gene expression of myogenic regulatory genes was measured by Real-Time PCR. Our results demonstrate that CEME, independent of the culture surface, was the main factor influencing the intense differentiation of muscle progenitor cells. The present results, for the first time, clearly demonstrated that the cultured tissue-like structure was capable of inducing contractions without externally applied impulses. It has been indicated that a small amount of CEME in media (about 1%) allows the culture of pseudo-tissue muscle capable of spontaneous contraction. The study showed that the graphene oxide may be used as a niche for differentiating muscle cells, but the decisive influence on the maturation of muscle tissue, especially muscle contractions, depends on the complexity of the applied growth factors.

Human dihydrofolate reductase is a substrate of protein kinase CK2α.

Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial, antiprotozoan, and immunosuppressive chemotherapies, and CK2 protein kinase is an ubiquitous enzyme involved in many processes, such as tRNA and rRNA synthesis, apoptosis, cell cycle or oncogenic transformation. We show for the first time that CK2α subunit strongly interacted with and phosphorylated DHFR in vitro. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we determined DHFR-CK2α binding kinetic parameters (Kd below 0.5 µM, kon = 10.31 × 104 M-1s-1 and koff = 1.40 × 10-3s-1) and calculated Gibbs free energy (-36.4 kJ/mol). In order to identify phosphorylation site(s) we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to CK2α subunit catalytic activity and the results pointed to serine 168 as a phosphorylation site. Mass spectrometry analyses confirmed the presence of phosphoserine 168 and revealed additionally the presence of phosphoserine 145, although the latter phosphorylation was on a very low level.

A short splicing isoform of HBS1L links the cytoplasmic exosome and SKI complexes in humans.

The exosome complex is a major eukaryotic exoribonuclease that requires the SKI complex for its activity in the cytoplasm. In yeast, the Ski7 protein links both complexes, whereas a functional equivalent of the Ski7 has remained unknown in the human genome. Proteomic analysis revealed that a previously uncharacterized short splicing isoform of HBS1L (HBS1LV3) is the long-sought factor linking the exosome and SKI complexes in humans. In contrast, the canonical HBS1L variant, HBS1LV1, which acts as a ribosome dissociation factor, does not associate with the exosome and instead interacts with the mRNA surveillance factor PELOTA. Interestingly, both HBS1LV1 and HBS1LV3 interact with the SKI complex and HBS1LV1 seems to antagonize SKI/exosome supercomplex formation. HBS1LV3 contains a unique C-terminal region of unknown structure, with a conserved RxxxFxxxL motif responsible for exosome binding and may interact with the exosome core subunit RRP43 in a way that resembles the association between Rrp6 RNase and Rrp43 in yeast. HBS1LV3 or the SKI complex helicase (SKI2W) depletion similarly affected the transcriptome, deregulating multiple genes. Furthermore, half-lives of representative upregulated mRNAs were increased, supporting the involvement of HBS1LV3 and SKI2W in the same mRNA degradation pathway, essential for transcriptome homeostasis in the cytoplasm.

Exonuclease hDIS3L2 specifies an exosome-independent 3'-5' degradation pathway of human cytoplasmic mRNA.

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5'-3' exoribonuclease hXRN1 and the 3'-5' exoribonucleolytic RNA exosome complex. However, in addition to the exosome-associated 3'-5' exonucleases hDIS3 and hDIS3L, the human genome encodes another RNase II/R domain protein-hDIS3L2. Here, we show that hDIS3L2 is an exosome-independent cytoplasmic mRNA 3'-5' exonuclease, which exhibits processive activity on structured RNA substrates in vitro. hDIS3L2 associates with hXRN1 in an RNA-dependent manner and can, like hXRN1, be found on polysomes. The impact of hDIS3L2 on cytoplasmic RNA metabolism is revealed by an increase in levels of cytoplasmic RNA processing bodies (P-bodies) upon hDIS3L2 depletion, which also increases half-lives of investigated mRNAs. Consistently, RNA sequencing (RNA-seq) analyses demonstrate that depletion of hDIS3L2, like downregulation of hXRN1 and hDIS3L, causes changed levels of multiple mRNAs. We suggest that hDIS3L2 is a key exosome-independent effector of cytoplasmic mRNA metabolism.

Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease.

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5' ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.

hUTP24 is essential for processing of the human rRNA precursor at site A1, but not at site A0.

Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5'-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site.

A new strategy for gene targeting and functional proteomics using the DT40 cell line.

DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry-based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination-based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.