RESUMEN
The steady-state levels of protein sumoylation depend on relative rates of conjugation and desumoylation. Whether SUMO modifications are generally long-lasting or short-lived is unknown. Here we show that treating budding yeast cultures with 1,10-phenanthroline abolishes most SUMO conjugations within one minute, without impacting ubiquitination, an analogous post-translational modification. 1,10-phenanthroline inhibits the formation of the E1~SUMO thioester intermediate, demonstrating that it targets the first step in the sumoylation pathway. SUMO conjugations are retained after treatment with 1,10-phenanthroline in yeast that express a defective form of the desumoylase Ulp1, indicating that Ulp1 is responsible for eliminating existing SUMO modifications almost instantly when de novo sumoylation is inhibited. This reveals that SUMO modifications are normally extremely transient because of continuous desumoylation by Ulp1. Supporting our findings, we demonstrate that sumoylation of two specific targets, Sko1 and Tfg1, virtually disappears within one minute of impairing de novo sumoylation. Altogether, we have identified an extremely rapid and potent inhibitor of sumoylation, and our work reveals that SUMO modifications are remarkably short-lived.
Asunto(s)
Fenantrolinas , Saccharomyces cerevisiae , Sumoilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
High yield purification of Ulp1 is required during the isolation and purification of SUMO-tagged recombinant proteins. However, when expressed as a soluble protein, Ulp1 is toxic to E. coli host cells and most of the protein forms inclusion bodies. The extraction of insoluble Ulp1 followed by its purification and refolding into its active form is a lengthy and costly procedure. In our present study, we developed a simple, cost effective procedure for the large scale production of active Ulp1 that can be used for industrial scale requirements.
Asunto(s)
Escherichia coli , Péptido Hidrolasas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Péptido Hidrolasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismoRESUMEN
BACKGROUND: Mammalian metallothioneins (MTs) are small (6-7 kDa), intracellular, cysteine-rich, metal-binding proteins involved, inter alia, in the homeostasis of zinc and copper, detoxification of heavy metals, antioxidation against reactive oxygen species, and protection against DNA damage. The high cysteine content (~ 30%) in MTs makes them toxic to bacterial cells during protein production, resulting in low yield. To address this issue, we present for the first time a combinatorial approach using the small ubiquitin-like modifier (SUMO) and/or sortase as fusion tags for high-level expression of human MT3 in E. coli and its purification by three different strategies. RESULTS: Three different plasmids were generated using SUMO, sortase A pentamutant (eSrtA), and sortase recognition motif (LPETG) as removable fusion tags for high-level expression and purification of human MT3 from the bacterial system. In the first strategy, SUMOylated MT3 was expressed and purified using Ulp1-mediated cleavage. In the second strategy, SUMOylated MT3 with a sortase recognition motif at the N-terminus of MT3 was expressed and purified using sortase-mediated cleavage. In the final strategy, the fusion protein His6-SUMO-eSrtA-LPETG-MT3 was expressed and purified by one-step sortase-mediated inducible on-bead autocleavage. Using these three strategies the apo-MT3 was purified in a yield of 11.5, 11, and 10.8 mg/L, respectively, which is the highest yield achieved for MT expression and purification to date. No effect of MT3 on Ni2+-containing resin was observed. CONCLUSION: The SUMO/sortase-based strategy used as the production system for MT3 resulted in a very high expression level and protein production yield. The apo-MT3 purified by this strategy contained an additional glycine residue and had similar metal binding properties as WT-MT3. This SUMO-sortase fusion system is a simple, robust, and inexpensive one-step purification approach for various MTs as well as other toxic proteins with very high yield via immobilized metal affinity chromatography (IMAC).
Asunto(s)
Calcio , Cisteína , Metalotioneína 3 , Humanos , Proteínas Bacterianas/genética , Escherichia coli/genética , Ubiquitina , Metalotioneína 3/metabolismoRESUMEN
Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (â¼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only â¼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.
Asunto(s)
Saccharomyces cerevisiae , Termotolerancia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sumoilación , Termotolerancia/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
The nuclear pore complex (NPC) provides a permeable barrier between the nucleoplasm and cytoplasm. In a subset of NPC constituents that regulate meiosis in the fission yeast Schizosaccharomyces pombe, we found that nucleoporin Nup132 (homolog of human Nup133) deficiency resulted in transient leakage of nuclear proteins during meiosis I, as observed in the nup132 gene-deleted mutant. The nuclear protein leakage accompanied the liberation of the small ubiquitin-like modifier (SUMO)-specific ubiquitin-like protease 1 (Ulp1) from the NPC. Ulp1 retention at the nuclear pore prevented nuclear protein leakage and restored normal meiosis in a mutant lacking Nup132. Furthermore, using mass spectrometry analysis, we identified DNA topoisomerase 2 (Top2) and RCC1-related protein (Pim1) as the target proteins for SUMOylation. SUMOylation levels of Top2 and Pim1 were altered in meiotic cells lacking Nup132. HyperSUMOylated Top2 increased the binding affinity at the centromeres of nup132 gene-deleted meiotic cells. The Top2-12KR sumoylation mutant was less localized to the centromeric regions. Our results suggest that SUMOylation of chromatin-binding proteins is regulated by the NPC-bound SUMO-specific protease and is important for the progression of meiosis.
Asunto(s)
Poro Nuclear , Schizosaccharomyces , Humanos , Poro Nuclear/metabolismo , Sumoilación , Schizosaccharomyces/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Meiosis , Péptido Hidrolasas/metabolismo , Ubiquitinas/genéticaRESUMEN
BACKGROUND: In plants, heterotrimeric G-protein (Gγ) subunits are diverse, and they have structural plasticity to provide functional selectivity to the heterotrimer. Although the Gß and Gγ subunits dimerize to function in the signaling pathway, the interaction mechanism of various Gγ subunits with the Gß subunit partners is still elusive. OBJECTIVE: To better understand the interaction mechanism, one approach is to separate the subunits for the re-assembly in vitro. Hence, developing a reliable method for achieving the efficient production and purification of these proteins has become necessary. METHODS: In this study, Gγ1 and Gγ2 proteins from Oryza sativa and Arabidopsis thaliana were successfully identified, cloned, expressed in bacteria, and purified as recombinant proteins with the fusion tags. Highly expressed recombinant Gγ subunits in E. coli were digested by proteases, which were also produced in the presented study. RESULTS: Preliminary structural characterization studies without the Gß partners showed that Gγ1 proteins have disordered structures with coiled-coil, α-helix extensions, and loops, whereas the Gγ2 protein has a more dominant ß-sheet and turns structure. Finally, computational analyses performed on Gγ genes have laid the foundation of new targets for biotechnological purposes. CONCLUSION: The proposed optimized expression and purification protocol can contribute to investigations on the Gßγ binding mechanism in plant G-protein signaling. The investigations on selective binding are critical to shed light on the role(s) of different plant Gγ subunit types in biological processes.
Asunto(s)
Arabidopsis , Proteínas de Unión al GTP Heterotriméricas , Arabidopsis/genética , Proteínas Portadoras/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Unión al GTP Heterotriméricas/química , Proteínas de Unión al GTP Heterotriméricas/genética , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas de Plantas/química , Plantas/metabolismoRESUMEN
The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. We describe cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. We also describe assays for Smo sumoylation in both Drosophila and mammalian cultured cells.
Asunto(s)
Sumoilación , Ubiquitinación , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Mamíferos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.
Asunto(s)
Reparación del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Cisteína Endopeptidasas/metabolismo , ADN/metabolismo , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación/genética , Sumoilación/fisiología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Intrinsically disordered proteins (IDPs) describe a group of proteins that do not have a regular tertiary structure and typically have very little ordered secondary structure. Despite not following the biochemical dogma of "structure determines function" and "function determines structure," IDPs have been identified as having numerous biological functions. We describe here the steps to express and purify the intrinsically disordered stress response protein, Late embryogenesis abundant protein 3-2 from Arabidopsis thaliana (AtLEA 3-2), with 15N and 13C isotopes in E. coli, although the protocol can be adapted for any IDP with or without isotopic labeling. The atlea 3-2 gene has been cloned into the pET-SUMO vector that in addition to the SUMO portion encodes an N-terminal hexahistidine sequence (His-tag). This vector allows for the SUMO-AtLEA 3-2 fusion protein to be purified using Ni-affinity chromatography and, through the use of ubiquitin-like-specific protease 1 (Ulp1, a SUMO protease), results in an AtLEA 3-2 with a native N-terminus. We also describe the expression and purification of Ulp1 itself.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/aislamiento & purificación , Proteínas Intrínsecamente Desordenadas/metabolismo , Fraccionamiento Celular , Electroforesis en Gel de Poliacrilamida , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The development of novel methods for highly efficient protein purification remains a research focus in the biotechnology field because conventional purification approaches, including affinity purification, gel filtration, and ion-exchange chromatography, require complex manipulation steps and are costly. Here, we describe a simple and rapid protein purification strategy in which the SUMO tag and Ulp1 protease are surface-displayed separately on Escherichia coli cells. After protein induction, the cells are harvested, resuspended in cleavage buffer, and incubated together for cleavage. In this approach, the surface-displayed Ulp1 cleaves the membrane-anchored SUMO fusion protein, resulting in the release of the target protein from the C-terminal of SUMO into the solution. The bacterial cells harboring SUMO and Ulp1 on their surfaces can be easily removed by centrifugation. To evaluate the purification method, we used red fluorescent protein (mCherry). Purified mCherry protein (7.72 ± 1.05 mg from 1 L of bacterial culture) was obtained after only 30 min of incubation. The protein purity was higher than 80%, and could be further improved (> 90%) by simple ultrafiltration. This study offers a promising and simple strategy for the purification of recombinant protein in its native form that requires only cleavage and centrifugation steps.
RESUMEN
In addition to their role in regulating transport across the nuclear envelope, increasing evidence suggests nuclear pore complexes (NPCs) function in regulating gene expression. For example, the induction of certain genes (e.g., yeast INO1) is accompanied by their movement from the nuclear interior to NPCs. As sumoylation has been linked to the regulation of chromatin spatial organization and transcriptional activity, we investigated the role of sumoylation in the expression and NPC recruitment of the INO1 gene. We observed that induction of INO1 is accompanied by both increased and decreased sumoylation of proteins associated with specific regions along the INO1 locus. Furthermore, we show that the E3 ligase Siz2/Nfi1 is required for targeting the INO1 locus to the NPC where it interacts with the SUMO isopeptidase Ulp1. Our data suggest that this interaction is required for both the association of INO1 with the NPC and for its normal expression. These results imply that sumoylation is a key regulator of INO1 targeting to the NPC, and a cycle of sumoylation and NPC-associated desumoylation events contribute to the regulation of INO1 expression.
RESUMEN
Brassinosteroids (BRs) play crucial roles in plant development, but little is known of mechanisms that integrate environmental cues into BR signaling. Conjugation to the small ubiquitin-like modifier (SUMO) is emerging as an important mechanism to transduce environmental cues into cellular signaling. In this study, we show that SUMOylation of BZR1, a key transcription factor of BR signaling, provides a conduit for environmental influence to modulate growth during stress. SUMOylation stabilizes BZR1 in the nucleus by inhibiting its interaction with BIN2 kinase. During salt stress, Arabidopsis plants arrest growth through deSUMOylation of BZR1 in the cytoplasm by promoting the accumulation of the BZR1 targeting SUMO protease, ULP1a. ULP1a mutants are salt tolerant and insensitive to the BR inhibitor, brassinazole. BR treatment stimulates ULP1a degradation, allowing SUMOylated BZR1 to accumulate and promote growth. This study uncovers a mechanism for integrating environmental cues into BR signaling to shape growth.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Brasinoesteroides/metabolismo , Cisteína Endopeptidasas/genética , Proteínas de Unión al ADN/genética , Transducción de Señal/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Núcleo Celular , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/metabolismo , SumoilaciónRESUMEN
In this study, we analyzed and compared the properties of yeast Ulp1 protease in active inclusion bodies (IBs) as special protein immobilizate, and the soluble Ulp1 via oriented immobilization. Fusion of the N-terminal self-assembling peptide GFIL8 to the Ulp1 increased production of active IBs in Escherichia coli. Attachment of the N-terminal cellulose-binding module facilitated the constructed protein immobilized on the regenerated amorphous cellulose (RAC) with a binding capacity up to about 235 mg protein per gram of RAC. Compared with the immobilized soluble construct, the insoluble Ulp1 showed higher resistance to limited proteolysis with trypsin digestion, lower leaky amount at different storage temperatures, but more rapid decrease in cleavage activity after stored at 4°C for 8 days. The immobilized soluble Ulp1 maintained about 42% initial cleavage activity with repetitive use successively, whereas the aggregated Ulp1 lost its cleavage capacity after cleaving the protein substrate once. Crosslinking of IBs mediated by glutaraldehyde inactivated the Ulp1. Freshly prepared and used IBs showed similar resistance to protease-K digestion, and comparable binding capacity of Congo red and thioflavin T. Taken together, due to different advantages, the Ulp1 constructs as carrier-free and carrier-dependent immobilizates are used under different conditions.
Asunto(s)
Cisteína Endopeptidasas , Enzimas Inmovilizadas , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Celulosa/química , Celulosa/metabolismo , Clonación Molecular , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Unión Proteica , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , SolubilidadRESUMEN
Heterochromatin in Saccharomyces cerevisiae is found at the telomeres and silent mating type loci. Many sub-telomeric loci are naturally silenced by this mechanism. In addition, when euchromatic genes are placed proximal to telomeric repeats they are subjected to heritable gene silencing that is referred to as telomere position effect. Establishment and maintenance of TPE is dependent on the assembly of silent information regulator proteins at these loci. Here we show that dosage of SUMO isopeptidase, Ulp1, is important for regulation of TPE. Moderate elevation of Ulp1 reduces silencing of both, the euchromatic gene placed proximal to telomeric repeats and the sub-telomeric genes that are silenced by TPE. We further demonstrate that this loss in silencing is due to reduced recruitment of one of the silent information regulators, Sir3p. We show that SUMO peptidase, Ulp1, regulates telomeric position effect by regulating the recruitment of Sir proteins.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telómero/metabolismo , Cisteína Endopeptidasas/genética , Proteínas de Unión al ADN/genética , Silenciador del Gen/fisiología , Heterocromatina/metabolismo , Heterocromatina/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Telómero/genética , Transactivadores , Transcripción GenéticaRESUMEN
At present, approximately 30% of eukaryotic proteins can be expressed in a soluble form in Escherichia coli. In this study, a pCold-SUMOa plasmid was constructed in order to express heterologous proteins fused with SUMO by a cold-shock expression vector. The human cysteine desulfurase NFS1 and a chimeric cysteine desulfurase namely, EH-IscS were successfully expressed in E. coli. The proteins were particularly difficult to be produced functionally, due to their readily sequestered nature. The recombinant cysteine desulfurases that were generated by pCold-SUMOa exhibited higher activity, solubility and stability compared with the well-known plasmid pCold I. In contrast to the pCold TF plasmid, the SUMO tag conferred no biological activity with regard to the conformation of the cysteine desulfurases. Furthermore, the SUMO protease 1 can efficiently recognize the tertiary structure of SUMO and cleave it. The data indicate that the pCold-SUMOa vector is a promising tool for native eukaryotic protein production.
RESUMEN
Ulp1 and Ulp2, in the yeast Saccharomyces cerevisiae, are the founding members of deSUMOylating enzymes. These enzymes remove small ubiquitin-like modifier (SUMO) from proteins and are conserved in all eukaryotes. Previous studies have shown that Ulp1 deSUMOylates the bulk of intracellular SUMOylated proteins, whereas Ulp2 is a highly specific enzyme. However, the mechanism for Ulp2's substrate specificity has been insufficiently understood. Here we show that the C-terminal regulatory domain of Ulp2 contains three distinct, yet conserved, motifs that control its in vivo substrate specificity and cell growth. Among them, a SUMO-interacting motif (SIM) was found to coordinate with the domain of Ulp2 that binds to the nucleolar protein Csm1 to ensure maximal deSUMOylation of Ulp2's nucleolar substrates. We found that whereas the Csm1-binding domain of Ulp2 recruits this enzyme to the nucleolus, Ulp2's C-terminal SIM promotes its SUMO protease activity and plays a key role in mediating the in vivo specificity of Ulp2. Thus, the substrate specificity of Ulp2 is controlled by both its subcellular localization and the SUMOylation status of its substrates. These findings illustrate the highly coordinated and dynamic nature of the SUMO pathways in maintaining homeostasis of intracellular SUMOylation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Endopeptidasas/química , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Regulación Fúngica de la Expresión Génica , Cinética , Proteínas Nucleares/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad por Sustrato , Sumoilación , Ubiquitina/genética , UbiquitinaciónRESUMEN
OBJECTIVE: To fabricate an active and stable enzyme through covalent immobilization, a Ubl-specific protease (Ulp1) was used to cleave small ubiquitin-like modifier (SUMO) fusion proteins. RESULTS: We immobilized Ulp1 on N-hydroxysuccinimide (NHS)-activated Sepharose with a coupling efficiency of 1.7 mg/ml. The immobilized Ulp1 maintains 95% substrate-cleavage ability and significantly enhances pH and thermal stability, especially can withstand pH of 10.5. Besides resistance against some small molecules, the immobilized Ulp1 can tolerate 15% (v/v) DMSO and 20% (v/v) ethanol. It can be reused for more than 15 batch reactions with 90% activity retention. This provides a fast purification system to quickly obtain cleaved recombinant proteins with 95% purity from cell lysates with the application of immobilized Ulp1. CONCLUSIONS: Ulp1 used in immobilization form is a potentially useful tool for cleavage of SUMO-tagged proteins and may reduce time and cost of protein purification.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Sefarosa , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Cisteína Endopeptidasas/química , Estabilidad de Enzimas , Enzimas Inmovilizadas/química , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.
Asunto(s)
Cisteína Endopeptidasas/genética , Proteína SUMO-1/genética , Animales , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/toxicidad , Plaquetas/efectos de los fármacos , Bothrops , Venenos de Crotálidos/genética , Venenos de Crotálidos/toxicidad , Cisteína Endopeptidasas/metabolismo , Desintegrinas/genética , Desintegrinas/toxicidad , Escherichia coli/genética , Humanos , Fosfolipasa D/genética , Fosfolipasa D/toxicidad , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/toxicidad , Proteínas Recombinantes de Fusión/toxicidad , Proteína SUMO-1/metabolismo , Venenos de Araña , ArañasRESUMEN
Protein sumoylation is a reversible posttranslational modification that controls multiple processes during cell cycle progression. Frequently, SUMO synergistically targets various subunits in a protein complex to modulate its function, leading to what has been defined as protein group sumoylation. Different subunits in the RENT (regulator of nucleolar silencing and telophase) complex, including Net1, Sir2, and Cdc14, can be coupled to SUMO, making it difficult to ascertain the role of this modification. Here we describe a method to downregulate sumoylation in RENT, consisting in the fusion of a catalytic domain of the Ulp1 SUMO protease (Ulp Domain; UD) to the C-terminus of members in the complex using epitope tags as linkers. Targeting of the UD to specific loci can be simplified by transformation of PCR-amplified cassettes. The presence of the UD in the complex allows the concurrent downregulation of sumoylated species in the RENT complex, what can be easily monitored by pull-down of SUMO conjugates. This methodology can be applied to other protein complexes exhibiting group sumoylation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Sirtuina 2/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Proteínas de Ciclo Celular/genética , Clonación Molecular/métodos , Cisteína Endopeptidasas/genética , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Mutagénesis Sitio-Dirigida/métodos , Proteínas Nucleares/genética , Reacción en Cadena de la Polimerasa/métodos , Proteínas Tirosina Fosfatasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Sirtuina 2/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación , Transformación GenéticaRESUMEN
The budding yeast small ubiquitin-like modifier (SUMO) protease Ulp1p catalyzes both the processing of newly synthesized SUMO to its mature form and the deconjugation of SUMO from target proteins, thereby regulating a wide range of cellular processes including cell division, DNA repair, DNA replication, transcription, and mRNA quality control. Ulp1p is localized primarily at the nuclear pore complex (NPC) through interactions involving the karyopherins Kap121p and Kap95p-Kap60p heterodimer and a subset of nuclear pore-associated proteins. The sequestration of Ulp1p at the nuclear periphery is crucial for the proper control of protein desumoylation. To gain insights into the role of the karyopherins in regulating the localization of Ulp1p, we have determined the crystal structures of Kap121p and Kap60p bound to the N-terminal non-catalytic domain of Ulp1p that is necessary and sufficient for NPC targeting. Contrary to a previous proposal that Ulp1p is tethered to the transport channel of the NPC through unconventional interactions with the karyopherins, our structures reveal that Ulp1p has canonical nuclear localization signals (NLSs): (1) an isoleucine-lysine-NLS (residues 51-55) that binds to the NLS-binding site of Kap121p, and (2) a classical bipartite NLS (residues 154-172) that binds to the major and minor NLS-binding sites of Kap60p. Ulp1p also binds Kap95p directly, and the Ulp1p-Kap95p binding is enhanced by the importin-ß-binding domain of Kap60p. GTP-bound Gsp1p (the yeast Ran ortholog) and the exportin Cse1p cooperate to release Ulp1p from the karyopherins, indicating that the stable sequestration of Ulp1p to the NPC would require a karyopherin-independent mechanism to anchor Ulp1p at the NPC.