Ubiquitin-chains dynamics and its role regulating crucial cellular processes.

The proteome adapts to multiple situations occurring along the life of the cell. To face these continuous changes, the cell uses posttranslational modifications (PTMs) to control the localization, association with multiple partners, stability, and activity of protein targets. One of the most dynamic protein involved in PTMs is Ubiquitin (Ub). Together with other members of the same family, known as Ubiquitin-like (UbL) proteins, Ub rebuilds the architecture of a protein in a few minutes to change its properties in a very efficient way. This capacity of Ub and UbL is in part due to their potential to form complex architectures when attached to target proteins or when forming Ub chains. The highly dynamic formation and remodeling of Ub chains is regulated by the action of conjugating and deconjugating enzymes that determine, in due time, the correct chain architecture for a particular cellular function. Chain remodeling occurs in response to physiologic stimuli but also in pathologic situations. Here, we illustrate well-documented cases of chain remodeling during DNA repair, activation of the NF-κB pathway and autophagy, as examples of this dynamic regulation. The crucial role of enzymes and cofactors regulating chain remodeling is discussed.

Deep Eutectic Solvents as New Reaction Media to Produce Alkyl-Glycosides Using Alpha-Amylase from Thermotoga maritima.

Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.

Analysis of PTEN ubiquitylation and SUMOylation using molecular traps.

The function of the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is tightly controlled by post-translational modifications (PTMs) including ubiquitin or Small Ubiquitin-related MOdifiers (SUMO). It is known that SUMOylation by SUMO-1, SUMO-2/-3, mono- or polyubiquitylation have a distinct impact on PTEN activity, localisation and/or stability, however the molecular mechanisms governing these processes are still unclear. Studying PTM regulated events has always been a difficult task due to their labile nature. Here, we propose an update on the role of these PTMs on PTEN function, as well as a methodological overview on the use of molecular traps named SUMO Binding Entities (SUBEs) or Tandem Ubiquitin Binding Entities (TUBEs) to capture SUMOylated or Ubiquitylated forms of PTEN respectively. When combined with in vitro SUMOylation or Ubiquitylation assays, the use of molecular traps facilitate the detection of modified forms of PTEN. SUMO and ubiquitin-traps are also suitable to capture endogenously modified forms of PTEN after expression of E3 ligases or treatment with chemical inhibitors. This versatile approach represents an interesting alternative to explore PTEN regulation by SUMO and ubiquitin under physiological or pathological conditions.

Development of two novel high-throughput assays to quantify ubiquitylated proteins in cell lysates: application to screening of new anti-malarials.

BACKGROUND: The ubiquitin proteasome system (UPS) is one of the main proteolytical pathways in eukaryotic cells and plays an essential role in key cellular processes such as cell cycle, stress response, signal transduction, and transcriptional regulation. Many components of this pathway have been implicated in diverse pathologies including cancer, neurodegeneration and infectious diseases, such as malaria. The success of proteasome inhibitors in clinical trials underlines the potential of the UPS in drug discovery. METHODS: Plasmodium falciparum, the malaria causative pathogen, has been used to develop two assays that allow the quantification of the parasite protein ubiquitylation levels in a high-throughput format that can be used to find new UPS inhibitors. RESULTS: In both assays tandem ubiquitin binding entities (TUBEs), also known as ubiquitin traps, have been used to capture ubiquitylated proteins from cell lysates. The primary assay is based on AlphaLISA technology, and the orthogonal secondary assay relies on a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) system. A panel of well-known proteasome inhibitors has been used to validate both technologies. An excellent correlation was obtained between these biochemical assays and the standard whole cell assay that measures parasite growth inhibition. CONCLUSIONS: The two assays presented can be used in a high-throughput format to find new UPS inhibitors for P. falciparum and could help to identify new targets within this system. This methodology is also applicable to other cellular contexts or pathologies.

Breaking Bad Proteins-Discovery Approaches and the Road to Clinic for Degraders.

Proteolysis-targeting chimeras (PROTACs) describe compounds that bind to and induce degradation of a target by simultaneously binding to a ubiquitin ligase. More generally referred to as bifunctional degraders, PROTACs have led the way in the field of targeted protein degradation (TPD), with several compounds currently undergoing clinical testing. Alongside bifunctional degraders, single-moiety compounds, or molecular glue degraders (MGDs), are increasingly being considered as a viable approach for development of therapeutics, driven by advances in rational discovery approaches. This review focuses on drug discovery with respect to bifunctional and molecular glue degraders within the ubiquitin proteasome system, including analysis of mechanistic concepts and discovery approaches, with an overview of current clinical and pre-clinical degrader status in oncology, neurodegenerative and inflammatory disease.

Constitutive Activation of p62/Sequestosome-1-Mediated Proteaphagy Regulates Proteolysis and Impairs Cell Death in Bortezomib-Resistant Mantle Cell Lymphoma.

Protein ubiquitylation coordinates crucial cellular events in physiological and pathological conditions. A comparative analysis of the ubiquitin proteome from bortezomib (BTZ)-sensitive and BTZ-resistant mantle cell lymphoma (MCL) revealed an enrichment of the autophagy-lysosome system (ALS) in BTZ-resistant cells. Pharmacological inhibition of autophagy at the level of lysosome-fusion revealed a constitutive activation of proteaphagy and accumulation of proteasome subunits within autophagosomes in different MCL cell lines with acquired or natural resistance to BTZ. Inhibition of the autophagy receptor p62/SQSTM1 upon verteporfin (VTP) treatment disrupted proteaphagosome assembly, reduced co-localization of proteasome subunits with autophagy markers and negatively impacted proteasome activity. Finally, the silencing or pharmacological inhibition of p62 restored the apoptosis threshold at physiological levels in BTZ-resistant cells both in vitro and in vivo. In total, these results demonstrate for the first time a proteolytic switch from the ubiquitin-proteasome system (UPS) to ALS in B-cell lymphoma refractory to proteasome inhibition, pointing out a crucial role for proteaphagy in this phenomenon and paving the way for the design of alternative therapeutic venues in treatment-resistant tumors.

Analysis of defective protein ubiquitylation associated to adriamycin resistant cells.

DNA damage activated by Adriamycin (ADR) promotes ubiquitin-proteasome system-mediated proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR-resistant breast cancer MCF7 (MCF7ADR) cells, protein ubiquitylation is significantly reduced compared to the parental MCF7 cells. Here, we used tandem ubiquitin-binding entities (TUBEs) to analyze the ubiquitylation pattern observed in MCF7 or MCF7ADR cells. While in MCF7, the level of total ubiquitylation increased up to six-fold in response to ADR, in MCF7ADR cells only a two-fold response was found. To further explore these differences, we looked for cellular factors presenting ubiquitylation defects in MCF7ADR cells. Among them, we found the tumor suppressor p53 and its ubiquitin ligase, Mdm2. We also observed a drastic decrease of proteins known to integrate the TUBE-associated ubiquitin proteome after ADR treatment of MCF7 cells, like histone H2AX, HMGB1 or ß-tubulin. Only the proteasome inhibitor MG132, but not the autophagy inhibitor chloroquine partially recovers the levels of total protein ubiquitylation in MCF7ADR cells. p53 ubiquitylation is markedly increased in MCF7ADR cells after proteasome inhibition or a short treatment with the isopeptidase inhibitor PR619, suggesting an active role of these enzymes in the regulation of this tumor suppressor. Notably, MG132 alone increases apoptosis of MCF7ADR and multidrug resistant ovarian cancer A2780DR1 and A2780DR2 cells. Altogether, our results highlight the use of ubiquitylation defects to predict resistance to ADR and underline the potential of proteasome inhibitors to treat these chemoresistant cells.

A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation.

Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.

Real-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions Between SUMO Traps and Mono- or PolySUMO Moieties.

Isolating endogenous SUMOylated proteins is a challenging task due to the high reversibility of this posttranslational modification. We have shown that SUMO traps are useful tools for the enrichment and isolation of proteins modified by SUMO in vitro and in vivo. To characterize the affinity and specificity of different SUMO chains for these traps, that are based on SUMO-interacting motifs, we have used real-time surface plasmon resonance (SPR), which allows a label-free analysis of protein/protein interactions. Here, a protocol to determine the affinities of multivalent SUMO traps for polySUMO chains or mono-SUMO molecules by SPR is presented.

Analysis of SUMOylated Proteins in Cells and In Vivo Using the bioSUMO Strategy.

Posttranslational regulation of proteins by conjugation of ubiquitin- and ubiquitin-like molecules is a common theme in almost every known biological pathway. SUMO (small ubiquitin-related modifier) is dynamically added and deleted from many cellular substrates to control activity, localization, and recruitment of other SUMO-recognizing protein complexes. The dynamic nature of this modification and its low abundance in resting cells make it challenging to study, with susceptibility to deSUMOylases further complicating its analysis. Here we describe bioSUMO, a general method to isolate and analyze SUMOylated proteins from cultured cells, using Drosophila as a highlighted example. The method also has been validated in transgenic flies, as well as human cells. SUMOylated substrates are labeled by in vivo biotinylation, which facilitates their subsequent purification using streptavidin-based affinity chromatography under stringent conditions and with very low background. The bioSUMO approach can be used to validate whether a specific protein is modified, or used to analyze an entire SUMO subproteome. If coupled to quantitative proteomics methods, it may reveal how the SUMO landscape changes with different stimuli, or in diverse cell or tissue types. This technique offers a complementary approach to study SUMO biology and we expect that the strategy can be extended to other ubiquitin-like proteins.

Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs.

Tandem ubiquitin-binding entities (TUBEs) act as molecular traps to isolate polyubiquitylated proteins facilitating the study of this highly reversible posttranslational modification. We provide here sample preparation and adaptations required for TUBE-based enrichment of the ubiquitin proteome from tumor cell lines or primary cells. Our protocol is suitable to identify ubiquitin substrates, enzymes involved in the ubiquitin proteasome pathway, as well as proteasome subunits by mass spectrometry. This protocol was adapted to prepare affinity columns, reduce background, and improve the protein recovery depending on the sample source and necessities.

Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays.

Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels.

New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach.

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. BIOLOGICAL SIGNIFICANCE: In this work we analyze for the first time the interconnection between Plasmodium and human red blood cells ubiquitin-regulated proteins in the context of infection. We identified a number of human and Plasmodium proteins whose ubiquitylation pattern changes during the asexual infective stage. We demonstrate that ubiquitylation of REX1, a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, peaks in trophozoites stage. The ubiquitin-proteome from P. falciparum infected red blood cells (iRBCs) revealed a significant host-parasite crosstalk, underlining the importance of ubiquitin-regulated proteolytic activities during the intraerythrocytic developmental cycle (IDC) of P. falciparum. Major cellular processes defined from gene ontology such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection. Given the importance of ubiquitylation in the development of infectious diseases, this work provides a number of potential drug-target candidates that should be further explored.

The potential to target CCL5/CCR5 in breast cancer.

INTRODUCTION: Chemokines play a crucial role in breast cancer tumorigenesis and progression. Recently, the chemokine (C-C motif) ligand 5 (CCL5), which can be secreted either by tumor cells or by mesenchymal stromal cells recruited to the tumor, has been identified as a key node in the bidirectional communication between breast cancer and normal cells. AREAS COVERED: In this review, the authors discuss the role of CCL5/chemokine receptor 5 (CCR5) axis in promoting breast cancer onset and progression. Interrogation of large clinical databases has demonstrated increased expression of the CCL5/CCR5 axis in specific subtypes of breast cancer. The activation of the receptor CCR5 in breast cancer cells controls their invasiveness serving as a driver for metastasis. Furthermore, the CCL5/CCR5 axis participates in the recruitment of specific immune cells into tumors, inducing local immunosuppression and favoring tumor progression. EXPERT OPINION: The role of CCR5 in HIV infection led to the development of specific and potent CCR5 antagonists. The data reviewed here includes basic and translational studies that support the use of such CCR5 antagonists in breast cancer patients as adjuvant therapy to block the metastasis.

Targeting the ubiquitin proteasome system: beyond proteasome inhibition.

The Ubiquitin-Proteasome System (UPS) has been considered as privileged pharmacological target for drug development due to the tremendous potential for intervention on multiple pathologies including cancer, neurodegenerative diseases, immune diseases and multiple infections. The pharmacological potential of the UPS was revealed after the unpredicted success of proteasome inhibitors for the treatment of some haematological malignancies. After a decade of clinical use of bortezomib, this review summarizes part of the learned experience and recent advances on the development of alternative inhibitors of the UPS. A new generation of inhibitors, including those targeting subsets of proteasomes, are under investigation and it is likely that some of them will reach clinical trials. Beyond the proteasome inhibition, there are also other targets that can be blocked to attain directly or indirectly the UPS system. The ubiquitylation status of protein substrates is intimately linked to other post-translational modifications of the ubiquitin family, increasing the number of potential targets for clinical intervention. In addition to the obvious subsets of ubiquitin-conjugating and de-conjugating enzymes, a group of enzymatic activities regulating SUMOylation or NEDDylation have a potential impact on the activity of the UPS. The novel strategies explore the active site of those enzymes and/or the target recognition surfaces. The first inhibitors of these parallel pathways appeared to tackle a limited number of protein targets playing important roles on diverse pathologies. Although, a large majority of them have not yet been tested in clinical trials, the new inhibitors are expected to have fewer side effects than proteasome inhibitors.

Analysis of SUMOylated proteins using SUMO-traps.

SUMO-modified proteins are recognized by SUMO interacting motifs (SIMs), thus triggering diverse cellular responses. Here SIMs were used to develop SUMO-traps to capture endogenous SUMOylated proteins. Our results show that these small peptides are transferable motifs that maintain their SUMO binding capacity when fused to the heterologous carrier protein GST. The tandem disposition of SIMs increases the binding capacity of SUMO-traps to specifically interact with polySUMO but not poly-Ubiquitin chains. We demonstrate that this SUMO capturing system purifies SUMOylated proteins such as IκBα, PTEN, PML or p53 in vitro and in vivo. These properties can be used to explore the many critical functions regulated by protein SUMOylation.

Mycobacterium tuberculosis glycoproteomics based on ConA-lectin affinity capture of mannosylated proteins.

A Mycobacterium tuberculosis culture filtrate enriched with mannose-containing proteins was resolved by 2-DE gel. After ConA ligand blotting, 41 proteins were identified by mass spectrometry as putative glycoproteins with 34 of them new probably mannosylated proteins. These results contribute to the construction of the ConA affinity glycoprotein database of M. tuberculosis, and provide useful information for understanding the biological role of glycoproteins in mycobacteria.

Identification of novel bacterial plasminogen-binding proteins in the human pathogen Mycobacterium tuberculosis.

Binding and activation of human plasminogen (Plg) to generate the proteolytic enzyme plasmin (Plm) have been associated with the invasive potential of certain bacteria. In this work, proteomic analysis together with ligand blotting assays identified several major Plg-binding spots in Mycobacterium tuberculosis soluble extracts (SEs) and culture filtrate proteins. The identity of 15 different proteins was deduced by N-terminal and/or MS and corresponded to DnaK, GroES, GlnA1, Ag85 complex, Mpt51, Mpt64, PrcB, MetK, SahH, Lpd, Icl, Fba, and EF-Tu. Binding of Plg to recombinant M. tuberculosis DnaK, GlnA1, and Ag85B was further confirmed by ELISA and ligand blotting assays. The binding was inhibited by epsilon-aminocaproic acid, indicating that the interaction involved lysine residues. Plg bound to recombinant mycobacterial proteins was activated to Plm by tissue-type Plg activator. In contrast with recombinant proteins, M. tuberculosis SE enhanced several times the Plg activation mediated by the activator. Interestingly, GlnA1 was able to bind the extracellular matrix (ECM) protein fibronectin. Together these results show that M. tuberculosis posses several Plg receptors suggesting that bound Plg to bacteria surface, can be activated to Plm, endowing bacteria with the ability to break down ECM and basal membranes proteins contributing to tissue injury in tuberculosis.