Studying the ubiquitin code through biotin-based labelling methods.

Post-translational modifications of cellular substrates by members of the ubiquitin (Ub) and ubiquitin-like (UbL) family are crucial for regulating protein homeostasis in organisms. The term "ubiquitin code" encapsulates how this diverse family of modifications, via adding single UbLs or different types of UbL chains, leads to specific fates for substrates. Cancer, neurodegeneration and other conditions are sometimes linked to underlying errors in this code. Studying these modifications in cells is particularly challenging since they are usually transient, scarce, and compartment-specific. Advances in the use of biotin-based methods to label modified proteins, as well as their proximally-located interactors, facilitate isolation and identification of substrates, modification sites, and the enzymes responsible for writing and erasing these modifications, as well as factors recruited as a consequence of the substrate being modified. In this review, we discuss site-specific and proximity biotinylation approaches being currently applied for studying modifications by UbLs, highlighting the pros and cons, with mention of complementary methods when possible. Future improvements may come from bioengineering and chemical biology but even now, biotin-based technology is uncovering new substrates and regulators, expanding potential therapeutic targets to manipulate the Ub code.

Biotin-Based Strategies to Explore the World of Ubiquitin and Ubiquitin-Like Modifiers.

A complex code of cellular signals is mediated by ubiquitin and ubiquitin-like (Ub/UbL) modifications on substrate proteins. The so-called Ubiquitin Code specifies protein fates, such as stability, subcellular localization, functional activation or suppression, and interactions. Hundreds of enzymes are involved in placing and removing Ub/UbL on thousands of substrates, while the consequences of modifications and the mechanisms of specificity are still poorly defined. Challenges include rapid and transient engagement of enzymes and Ub/UbL interactors, low stoichiometry of modified versus non-modified cellular substrates, and protease-mediated loss of Ub/UbL in lysates. To decipher this complexity and confront the challenges, many tools have been created to trap and identify substrates and interactors linked to Ub/UbL modification. This review focuses on an assortment of biotin-based tools developed for this purpose (for example BioUbLs, UbL-ID, BioE3, BioID), taking advantage of the strong affinity of biotin-streptavidin and the stringent lysis/washing approach allowed by it, paired with sensitive mass-spectrometry-based proteomic methods. Knowing how substrates change during development and disease, the consequences of substrate modification, and matching substrates to particular UbL-ligating enzymes will contribute new insights into how Ub/UbL signaling works and how it can be exploited for therapies.

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

BACKGROUND: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.

PROTEOSTASIS: A European Network to Break Barriers and Integrate Science on Protein Homeostasis.

Protein homeostasis (proteostasis) is at the core of cellular functions. The European network PROTEOSTASIS was created to steer research and foster collaborations in the interconnected fields of posttranslational modifications by ubiquitin family members and protein turnover by proteasome, autophagy, and lysosomal systems in health and diseases, across the kingdoms of life.

Using Ubiquitin Binders to Decipher the Ubiquitin Code.

Post-translational modifications (PTMs) by ubiquitin (Ub) are versatile, highly dynamic, and involved in nearly all aspects of eukaryote biological function. The reversibility and heterogeneity of Ub chains attached to protein substrates have complicated their isolation, quantification, and characterization. Strategies have emerged to isolate endogenous ubiquitylated targets, including technologies based on the use of Ub-binding peptides, such as tandem-repeated Ub-binding entities (TUBEs). TUBEs allow the identification and characterization of Ub chains, and novel substrates for deubiquitylases (DUBs) and Ub ligases (E3s). Here we review their impact on purification, analysis of pan or chain-selective polyubiquitylated proteins and underline the biological relevance of this information. Together with peptide aptamers and other Ub affinity-based approaches, TUBEs will contribute to unraveling the secrets of the Ub code.

Impaired proteostasis in rare neurological diseases.

Rare diseases are classified as such when their prevalence is 1:2000 or lower, but even if each of them is so infrequent, altogether more than 300 million people in the world suffer one of the ∼7000 diseases considered as rare. Over 1200 of these disorders are known to affect the brain or other parts of our nervous system, and their symptoms can affect cognition, motor function and/or social interaction of the patients; we refer collectively to them as rare neurological disorders or RNDs. We have focused this review on RNDs known to have compromised protein homeostasis pathways. Proteostasis can be regulated and/or altered by a chain of cellular mechanisms, from protein synthesis and folding, to aggregation and degradation. Overall, we provide a list comprised of above 215 genes responsible for causing more than 170 distinct RNDs, deepening on some representative diseases, including as well a clinical view of how those diseases are diagnosed and dealt with. Additionally, we review existing methodologies for diagnosis and treatment, discussing the potential of specific deubiquitinating enzyme inhibition as a future therapeutic avenue for RNDs.

Truncated SALL1 Impedes Primary Cilia Function in Townes-Brocks Syndrome.

Townes-Brocks syndrome (TBS) is characterized by a spectrum of malformations in the digits, ears, and kidneys. These anomalies overlap those seen in a growing number of ciliopathies, which are genetic syndromes linked to defects in the formation or function of the primary cilia. TBS is caused by mutations in the gene encoding the transcriptional repressor SALL1 and is associated with the presence of a truncated protein that localizes to the cytoplasm. Here, we provide evidence that SALL1 mutations might cause TBS by means beyond its transcriptional capacity. By using proximity proteomics, we show that truncated SALL1 interacts with factors related to cilia function, including the negative regulators of ciliogenesis CCP110 and CEP97. This most likely contributes to more frequent cilia formation in TBS-derived fibroblasts, as well as in a CRISPR/Cas9-generated model cell line and in TBS-modeled mouse embryonic fibroblasts, than in wild-type controls. Furthermore, TBS-like cells show changes in cilia length and disassembly rates in combination with aberrant SHH signaling transduction. These findings support the hypothesis that aberrations in primary cilia and SHH signaling are contributing factors in TBS phenotypes, representing a paradigm shift in understanding TBS etiology. These results open possibilities for the treatment of TBS.

Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome.

Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research.

Regulation of the Ebola Virus VP24 Protein by SUMO.

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione S-transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.IMPORTANCE The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.

The role of SUMOylation during development.

During the development of multicellular organisms, transcriptional regulation plays an important role in the control of cell growth, differentiation and morphogenesis. SUMOylation is a reversible post-translational process involved in transcriptional regulation through the modification of transcription factors and through chromatin remodelling (either modifying chromatin remodelers or acting as a 'molecular glue' by promoting recruitment of chromatin regulators). SUMO modification results in changes in the activity, stability, interactions or localization of its substrates, which affects cellular processes such as cell cycle progression, DNA maintenance and repair or nucleocytoplasmic transport. This review focuses on the role of SUMO machinery and the modification of target proteins during embryonic development and organogenesis of animals, from invertebrates to mammals.

Multi-Omics Integration Highlights the Role of Ubiquitination in CCl4-Induced Liver Fibrosis.

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in chronic liver disease. Ubiquitination is a post-translational modification that is crucial for a plethora of physiological processes. Even though the ubiquitin system has been implicated in several human diseases, the role of ubiquitination in liver fibrosis remains poorly understood. Here, multi-omics approaches were used to address this. Untargeted metabolomics showed that carbon tetrachloride (CCl4)-induced liver fibrosis promotes changes in the hepatic metabolome, specifically in glycerophospholipids and sphingolipids. Gene ontology analysis of public deposited gene array-based data and validation in our mouse model showed that the biological process "protein polyubiquitination" is enriched after CCl4-induced liver fibrosis. Finally, by using transgenic mice expressing biotinylated ubiquitin (bioUb mice), the ubiquitinated proteome was isolated and characterized by mass spectrometry in order to unravel the hepatic ubiquitinated proteome fingerprint in CCl4-induced liver fibrosis. Under these conditions, ubiquitination appears to be involved in the regulation of cell death and survival, cell function, lipid metabolism, and DNA repair. Finally, ubiquitination of proliferating cell nuclear antigen (PCNA) is induced during CCl4-induced liver fibrosis and associated with the DNA damage response (DDR). Overall, hepatic ubiquitome profiling can highlight new therapeutic targets for the clinical management of liver fibrosis.

Cluster Locator, online analysis and visualization of gene clustering.

Summary: Genes sharing functions, expression patterns or quantitative traits are not randomly distributed along eukaryotic genomes. In order to study the distribution of genes that share a given feature, we present Cluster Locator, an online analysis and visualization tool. Cluster Locator determines the number, size and position of all the clusters formed by the protein-coding genes on a list according to a given maximum gap, the percentage of gene clustering of the list and its statistical significance. The output includes a visual representation of the distribution of genes and gene clusters along the reference genome. Availability and implementation: Cluster Locator is freely available at http://clusterlocator.bnd.edu.uy/. Supplementary information: Supplementary data are available at Bioinformatics online.

The Spalt Transcription Factors Generate the Transcriptional Landscape of the Drosophila melanogaster Wing Pouch Central Region.

The Drosophila genes spalt major (salm) and spalt-related (salr) encode Zn-finger transcription factors regulated by the Decapentaplegic (Dpp) signalling pathway in the wing imaginal disc. The function of these genes is required for cell survival and proliferation in the central region of the wing disc, and also for vein patterning in the lateral regions. The identification of direct Salm and Salr target genes, and the analysis of their functions, are critical steps towards understanding the genetic control of growth and patterning of the Drosophila wing imaginal disc by the Dpp pathway. To identify candidate Salm/Salr target genes, we have compared the expression profile of salm/salr knockdown wing discs with control discs in microarray experiments. We studied by in situ hybridization the expression pattern of the genes whose mRNA levels varied significantly, and uncovered a complex transcription landscape regulated by the Spalt proteins in the wing disc. Interestingly, candidate Salm/Salr targets include genes which expression is turned off and genes which expression is positively regulated by Salm/Salr. Furthermore, loss-of-function phenotypic analysis of these genes indicates, for a fraction of them, a requirement for wing growth and patterning. The identification and analysis of candidate Salm/Salr target genes opens a new avenue to reconstruct the genetic structure of the wing, linking the activity of the Dpp pathway to the development of this epithelial tissue.

Evolution of SUMO Function and Chain Formation in Insects.

SUMOylation, the covalent binding of Small Ubiquitin-like Modifier (SUMO) to target proteins, is a posttranslational modification that regulates critical cellular processes in eukaryotes. In insects, SUMOylation has been studied in holometabolous species, particularly in the dipteran Drosophila melanogaster, which contains a single SUMO gene (smt3). This has led to the assumption that insects contain a single SUMO gene. However, the analysis of insect genomes shows that basal insects contain two SUMO genes, orthologous to vertebrate SUMO1 and SUMO2/3. Our phylogenetical analysis reveals that the SUMO gene has been duplicated giving rise to SUMO1 and SUMO2/3 families early in Metazoan evolution, and that later in insect evolution the SUMO1 gene has been lost after the Hymenoptera divergence. To explore the consequences of this loss, we have examined the characteristics and different biological functions of the two SUMO genes (SUMO1 and SUMO3) in the hemimetabolous cockroach Blattella germanica and compared them with those of Drosophila Smt3. Here, we show that the metamorphic role of the SUMO genes is evolutionary conserved in insects, although there has been a regulatory switch from SUMO1 in basal insects to SUMO3 in more derived ones. We also show that, unlike vertebrates, insect SUMO3 proteins cannot form polySUMO chains due to the loss of critical lysine residues within the N-terminal part of the protein. Furthermore, the formation of polySUMO chains by expression of ectopic human SUMO3 has a deleterious effect in Drosophila. These findings contribute to the understanding of the functional consequences of the evolution of SUMO genes.

Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.

Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10.

Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. We have now devised a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, we find direct ubiquitination by Ube3a of three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as of the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, we report the genetic interaction in vivo between Ube3a and the C-terminal part of Rpn10. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells.

Integration of RNA processing and expression level control modulates the function of the Drosophila Hox gene Ultrabithorax during adult development.

Although most metazoan genes undergo alternative splicing, the functional relevance of the majority of alternative splicing products is still unknown. Here we explore this problem in the Drosophila Hox gene Ultrabithorax (Ubx). Ubx produces a family of six protein isoforms through alternative splicing. To investigate the functional specificity of the Ubx isoforms, we studied their role during the formation of the Drosophila halteres, small dorsal appendages that are essential for normal flight. Our work shows that isoform Ia, which is encoded by all Ubx exons, is more efficient than isoform IVa, which lacks the amino acids coded by two small exons, in controlling haltere development and regulating Ubx downstream targets. However, our experiments also demonstrate that the functional differences among the Ubx isoforms can be compensated for by increasing the expression levels of the less efficient form. The analysis of the DNA-binding profiles of Ubx isoforms to a natural Ubx target, spalt, shows no major differences in isoform DNA-binding activities, suggesting that alternative splicing might primarily affect the regulatory capacity of the isoforms rather than their DNA-binding patterns. Our results suggest that to obtain distinct functional outputs during normal development genes must integrate the generation of qualitative differences by alternative splicing to quantitative processes affecting isoform protein expression levels.

A Genotyped Case of Townes-Brocks Syndrome with Absent Pulmonary Valve Syndrome from Turkey.

Townes-Brocks syndrome (TBS) is a rare syndrome characterized by triad of anal, ear, and thumb anomalies. Further malformations/anomalies include congenital heart diseases, foot malformations, sensorineural and/or conductive hearing impairment, genitourinary malformations, and anomalies of eye and nervous system. Definitive diagnosis for TBS is confirmed by molecular analysis for mutations in the SALL1 gene. Only one known case of TBS with absent pulmonary valve syndrome (APVS) has been previously described to our knowledge. Here, we report a newborn diagnosed with TBS with APVS and tetralogy of Fallot (TOF) who was found to carry the most common pathogenic SALL1 gene mutation c.826C > T (p.R276X), with its surgical repair and postoperative follow-up. To our knowledge, this is the first genotyped case of TBS from Turkey to date. TBS should be suspected in the presence of ear, anal, and thumb malformations in a neonate. If a patient with TBS and TOF-APVS needs preoperative ventilation within the first months of life, this implies prolonged postoperative intubation and increased risk of mortality.