Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates.

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

COVID-19 diagnosis by SARS-CoV-2 Spike protein detection in saliva using an ultrasensitive magneto-assay based on disposable electrochemical sensor.

The outbreak of the COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome of Coronavirus 2 (SARS-CoV-2), has fueled the search for diagnostic tests aiming at the control and reduction of the viral transmission. The main technique used for diagnosing the Coronavirus disease (COVID-19) is the reverse transcription-polymerase chain reaction (RT-PCR) technique. However, considering the high number of cases and the underlying limitations of the RT-PCR technique, especially with regard to accessibility and cost of the test, one does not need to overemphasize the need to develop new and less expensive testing techniques that can aid the early diagnosis of the disease. With that in mind, we developed an ultrasensitive magneto-assay using magnetic beads and gold nanoparticles conjugated to human angiotensin-converting enzyme 2 (ACE2) peptide (Gln24-Gln42) for the capturing and detection of SARS-CoV-2 Spike protein in human saliva. The technique applied involved the use of a disposable electrochemical device containing eight screen-printed carbon electrodes which allow the simultaneous analysis of eight samples. The magneto-assay exhibited an ultralow limit of detection of 0.35 ag mL-1 for the detection of SARS-CoV-2 Spike protein in saliva. The magneto-assay was tested in saliva samples from healthy and SARS-CoV-2-infected individuals. In terms of efficiency, the proposed technique - which presented a sensitivity of 100.0% and specificity of 93.7% for SARS-CoV-2 Spike protein-exhibited great similarity with the RT-PCR technique. The results obtained point to the application potential of this simple, low-cost magneto-assay for saliva-based point-of-care COVID-19 diagnosis.

Ruthenium(II) Phosphine/Mercapto Complexes: Their in Vitro Cytotoxicity Evaluation and Actions as Inhibitors of Topoisomerase and Proteasome Acting as Possible Triggers of Cell Death Induction.

In this paper, a series of new ruthenium complexes of the general formula [Ru(NS)(dpphpy)(dppb)]PF6 (Ru1-Ru3), where dpphpy = diphenyl-2-pyridylphosphine, NS ligands = 2-thiazoline-2-thiol (tzdt, Ru1), 2-mercaptopyrimidine (pySm, Ru2), and 4,6-diamino-2-mercaptopyrimidine (damp, Ru3), and dppb = 1,4-bis(diphenylphosphino)butane, were synthesized and characterized by elemental analysis, spectroscopic techniques (IR, UV/visible, and 1D and 2D NMR), and X-ray diffraction. In the characterization, the correlation between the phosphorus atoms and their respective aromatic hydrogen atoms of the compounds in the assignment stands outs, by 1H-31P HMBC experiments. The compounds show anticancer activities against A549 (lung) and MDA-MB-231 (breast) cancer cell lines, higher than the clinical drug cisplatin. All of the complexes are more cytotoxic against the cancer cell lines than against the MRC-5 (lung) and MCF-10A (breast) nontumorigenic human cell lines. For A549 tumor cells, cell cycle analysis upon treatment with Ru2 showed that it inhibits the mitotic phase because arrest was observed in the Sub-G1 phase. Additionally, the compound induces cell death by an apoptotic pathway in a dose-dependent manner, according to annexin V-PE assay. The multitargeted character of the compounds was investigated, and the biomolecules were DNA, topoisomerase IB, and proteasome, as well as the fundamental biomolecule in the pharmacokinetics of drugs, human serum albumin. The experimental results indicate that the complexes do not target DNA in the cells. At low concentrations, the compounds showed the ability to partially inhibit the catalytic activity of topoisomerase IB in the process of relaxation of the DNA plasmid. Among the complexes assayed in cultured cells, complex Ru3 was able to diminish the proteasomal chymotrypsin-like activity to a greater extent.

Expression of nuclear XIAP associates with cell growth and drug resistance and confers poor prognosis in breast cancer.

Evasion from apoptosis is one of the hallmarks of cancer. X-linked inhibitor of apoptosis protein (XIAP) is known to modulate apoptosis by inhibiting caspases and ubiquitinating target proteins. XIAP is mainly found at the cytoplasm, but recent data link nuclear XIAP to poor prognosis in breast cancer. Here, we generated a mutant form of XIAP with a nuclear localization signal (XIAPNLS-C-term) and investigated the oncogenic mechanisms associated with nuclear XIAP in breast cancer. Our results show that cells overexpressing XIAPΔRING (RING deletion) and XIAPNLS-C-term exhibited XIAP nuclear localization more abundantly than XIAPwild-type. Remarkably, overexpression of XIAPNLS-C-term, but not XIAPΔRING, conferred resistance to doxorubicin and increased cellular proliferative capacity. Interestingly, Survivin and c-IAP1 expression were not associated with XIAP oncogenic effects. However, NFκB expression and ubiquitination of K63, but not K48 chains, were increased following XIAPNLS-C-term overexpression, pointing to nuclear signaling transduction. Consistently, multivariate analysis revealed nuclear, but not cytoplasmic XIAP, as an independent prognostic factor in hormone receptor-negative breast cancer patients. Altogether, our findings suggest that nuclear XIAP confers poor outcome and RING-associated breast cancer growth and chemoresistance.

Fbxl17 is rearranged in breast cancer and loss of its activity leads to increased global O-GlcNAcylation.

In cancer, many genes are mutated by genome rearrangement, but our understanding of the functional consequences of this remains rudimentary. Here we report the F-box protein encoded by FBXL17 is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased ubiquitination activity. Loss of the LRRs also differentially affected Fbxl17 binding to its targets. Thus, genomic rearrangements in FBXL17 are likely to disrupt SCFFbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved in O-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevated O-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway.

Structural and functional studies of the Leishmania braziliensis SGT co-chaperone indicate that it shares structural features with HIP and can interact with both Hsp90 and Hsp70 with similar affinities.

Molecular chaperones and co-chaperones play an essential role in the life cycles of protozoa belonging to the genus Leishmania. The small glutamine-rich TPR-containing protein (SGT) is a co-chaperone that can be divided into three domains: N-terminal, tetratricopeptide (TPR) and C-terminal. The TPR domain is responsible for interactions with both Hsp70 and Hsp90; however, the mechanism of interaction and the functionality of SGT are unclear. In this context, we present the structural and functional characterization of Leishmania braziliensis SGT (LbSGT), aiming to elucidate how this co-chaperone interacts with the Hsp90/Hsp70 chaperone machinery. Structurally, the recombinant LbSGT behaves as an α-helical, multidomain and elongated dimer in solution. Despite their low amino acid sequence identity and similarity, LbSGT shares structural properties and domain organization with the Hsp70-interacting protein (HIP) co-chaperone. Functionally, LbSGT is a cognate protein in L. braziliensis promastigote cells and interacts indiscriminately, with similar affinities, with both Hsp90 and Hsp70 chaperones, capable of working as an adaptor protein. Sequence analysis indicates that LbSGT interacts via a dicarboxylate clamp, the same mechanism used by the Hsp90-Hsp70-organizing protein (HOP) co-chaperone. These results suggest that SGT can develop the same function as HOP but using the HIP structural scaffold.

DNA damage response signaling does not trigger redistribution of SAMHD1 to nuclear foci.

SAMHD1 (Sterile alpha motif and histidine-aspartic acid (HD) domain containing protein 1) is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase) that restricts viral replication in infected cells. This protein is also involved in DNA repair by assisting in DNA end resection by homologous recombination (HR) after DNA double-strand break (DSB) induction with camptothecin (CPT) or etoposide (ETO). We showed that a monoclonal anti-SAMHD1 antibody produced against the full-length protein detected an unspecific 50 kDa protein that colocalized with dot-like structures after CPT treatment in HeLa cells. In contrast, a polyclonal anti-SAMHD1 antibody raised against the N-terminus of this protein specifically detected SAMHD1, as shown in Jurkat, HAP1KO and HEK293T SAMHD1-siRNA cell lysates compared with their respective controls. Our findings showed that SAMHD1 is not localized in dot-like structures under DSB induction in HeLa cells.

FBXO25 regulates MAPK signaling pathway through inhibition of ERK1/2 phosphorylation.

The FBXO25 mediates degradation of ELK-1 and thus inhibits transcriptional activation of immediate early genes (iEG). Here we show that FBXO25 regulates yet another node of this signaling pathway, by decreasing MAPK/ERK activity. We show that induction of FBXO25 reduced ERK1/2 phosphorylation independently of MEK1/2. Accordingly, in HAP1 FBXO25 knockout cells (FBXO25KO), we observed that upon PMA treatment ERK1/2 was more active than in parental cells. An increase in cell proliferation under receptor mediated activation of the ERK signaling pathway in FBXO25KO cells was also observed. Taken together we show that FBXO25 functions as a negative regulator of MAPK signaling though the reduction of ERK1/2 activation.

Mining a differential sialotranscriptome of Rhipicephalus microplus guides antigen discovery to formulate a vaccine that reduces tick infestations.

BACKGROUND: Ticks cause massive damage to livestock and vaccines are one sustainable substitute for the acaricides currently heavily used to control infestations. To guide antigen discovery for a vaccine that targets the gamut of parasitic strategies mediated by tick saliva and enables immunological memory, we exploited a transcriptome constructed from salivary glands from all stages of Rhipicephalus microplus ticks feeding on genetically tick-resistant and susceptible bovines. RESULTS: Different levels of host anti-tick immunity affected gene expression in tick salivary glands; we thus selected four proteins encoded by genes weakly expressed in ticks attempting to feed on resistant hosts or otherwise abundantly expressed in ticks fed on susceptible hosts; these sialoproteins mediate four functions of parasitism deployed by male ticks and that do not induce antibodies in naturally infected, susceptible bovines. We then evaluated in tick-susceptible heifers an alum-adjuvanted vaccine formulated with recombinant proteins. Parasite performance (i.e. weight and numbers of females finishing their parasitic cycle) and titres of antigen-specific antibodies were significantly reduced or increased, respectively, in vaccinated versus control heifers, conferring an efficacy of 73.2%; two of the antigens were strong immunogens, rich in predicted T-cell epitopes and challenge infestations boosted antibody responses against them. CONCLUSION: Mining sialotranscriptomes guided by the immunity of tick-resistant hosts selected important targets and infestations boosted immune memory against salivary antigens.

Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two ß-arrestin-biased agonists.

G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with ß-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or ß-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known ß-arrestin-biased agonists, such as [Sar(1), Ile(4), Ile(8)]-AngII (SII), and [Sar(1), D-Ala(8)]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned ß-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between "G protein-dependent" and "ß-arrestin-dependent" signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of ß-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization "G protein-dependent" vs. "ß-arrestin-dependent" signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with distinct side effects.

The F-box protein FBXO25 promotes the proteasome-dependent degradation of ELK-1 protein.

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: effect of catalase overexpression.

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500µM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with ß-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and ß-oxidation of fatty acids.

Angiotensin-converting enzyme inhibition augments the expression of rat elastase-2, an angiotensin II-forming enzyme.

Mounting evidence suggest that tissue levels of angiotensin (ANG) II are maintained in animals submitted to chronic angiotensin-converting enzyme (ACE) inhibitor treatment. We examined the expression levels of transcripts for elastase-2, a chymostatin-sensitive serine protease identified as the alternative pathway for ANG II generation from ANG I in the rat vascular tissue and the relative role of ACE-dependent and -independent pathways in generating ANG II in the rat isolated carotid artery rings of spontaneously hypertensive rats (SHR) and Wistar normotensive rats (WNR) treated with enalapril for 7 days. Enalapril treatment decreased blood pressure of SHR only and resulted in significantly more elastase-2 mRNA expression in carotid artery of both enalapril-treated WNR and SHR. Captopril induced a comparable rightward shift of concentration-response curves to ANG I in vehicle and enalapril-treated rats, although this effect was of lesser magnitude in SHR group. Chymostatin induced a rightward shift of the dose response to ANG I in vehicle-treated and a decrease in maximal effect of 22% in enalapril-treated WNR group. Maximal response induced by ANG I was remarkably reduced by chymostatin in enalapril-treated SHR carotid artery (by 80%) compared with controls (by 23%). Our data show that chronic ACE inhibition was associated with augmented functional role of non-ACE pathway in generating ANG II and increased elastase-2 gene expression, suggesting that this protease may contribute as an alternative pathway for ANG II generation when ACE is inhibited in the rat vascular tissue.

Identification of FBXO25-interacting proteins using an integrated proteomics approach.

FBXO25 is one of the 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of s-phase-kinase associated protein 1, really interesting new gene-box 1, Cullin 1, and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FAND). Combining two-step affinity purification followed by MS with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, beta-actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FAND, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions.

FBXO25-associated nuclear domains: a novel subnuclear structure.

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.