In the moonlight: non-catalytic functions of ubiquitin and ubiquitin-like proteases.

Proteases that cleave ubiquitin or ubiquitin-like proteins (UBLs) are critical players in maintaining the homeostasis of the organism. Concordantly, their dysregulation has been directly linked to various diseases, including cancer, neurodegeneration, developmental aberrations, cardiac disorders and inflammation. Given their potential as novel therapeutic targets, it is essential to fully understand their mechanisms of action. Traditionally, observed effects resulting from deficiencies in deubiquitinases (DUBs) and UBL proteases have often been attributed to the misregulation of substrate modification by ubiquitin or UBLs. Therefore, much research has focused on understanding the catalytic activities of these proteins. However, this view has overlooked the possibility that DUBs and UBL proteases might also have significant non-catalytic functions, which are more prevalent than previously believed and urgently require further investigation. Moreover, multiple examples have shown that either selective loss of only the protease activity or complete absence of these proteins can have different functional and physiological consequences. Furthermore, DUBs and UBL proteases have been shown to often contain domains or binding motifs that not only modulate their catalytic activity but can also mediate entirely different functions. This review aims to shed light on the non-catalytic, moonlighting functions of DUBs and UBL proteases, which extend beyond the hydrolysis of ubiquitin and UBL chains and are just beginning to emerge.

Extracellular production of Ulp1403-621 in leaky E. coli and its application in antimicrobial peptide production.

Small ubiquitin-like modifier (SUMO) tag is widely used to promote soluble expression of exogenous proteins, which can then be cleaved by ubiquitin-like protease 1 (Ulp1) to obtain interested protein. But the application of Ulp1 in large-scale recombinant protein production is limited by complicated purification procedures and high cost. In this study, we describe an efficient and simple method of extracellular production of Ulp1403-621 using a leaky Escherichia coli BL21(DE3), engineered by deleting the peptidoglycan-associated outer membrane lipoprotein (pal) gene. Ulp1403-621 was successfully leaked into extracellular supernatant by the BL21(DE3)-Δpal strain after IPTG induction. The addition of 1% glycine increased the extracellular production of Ulp1403-621 approximately four fold. Moreover, extracellular Ulp1403-621 without purification had high activities for cleaving SUMO fusion proteins, and antimicrobial peptide pBD2 obtained after cleavage can inhibit the growth of Staphylococcus aureus. The specific activity of extracellular Ulp1403-621 containing 1 mM EDTA and 8 mM DTT reached 2.0 × 106 U/L. Another commonly used protease, human rhinovirus 3C protease, was also successfully secreted by leaky E. coli strains. In conclusion, extracellular production of tool enzymes is an attractive way for producing large-scale active recombinant proteins at a lower cost for pharmaceutical, industrial, and biotechnological applications. KEY POINTS: • First report of extracellular production of Ulp1403-621 in leaky Escherichia coli BL21(DE3) strain. • One percent glycine addition into cultivation medium increased the extracellular production of Ulp1403-621 approximately four fold. • The specific activity of extracellular Ulp1403-621 produced in this study reached 2.0 × 106 U/L.

Genome-based high-resolution mapping of fusarium wilt resistance in sweet basil.

Fusarium wilt of basil is a disease of sweet basil (Ocimum basilicum L.) plants caused by the fungus Fusarium oxysporum f. sp. basilici (FOB). Although resistant cultivars were released > 20 years ago, the underlying mechanism and the genes controlling the resistance remain unknown. We used genetic mapping to elucidate FOB resistance in an F2 population derived from a cross between resistant and susceptible cultivars. We performed genotyping by sequencing of 173 offspring and aligning the data to the sweet basil reference genome. In total, 23,411 polymorphic sites were detected, and a single quantitative trait locus (QTL) for FOB resistance was found. The confidence interval was < 600 kbp, harboring only 60 genes, including a cluster of putative disease-resistance genes. Based on homology to a fusarium resistance protein from wild tomato, we also investigated a candidate resistance gene that encodes a transmembrane leucine-rich repeat - receptor-like kinase - ubiquitin-like protease (LRR-RLK-ULP). Sequence analysis of that gene in the susceptible parent vs. the resistant parent revealed multiple indels, including an insertion of 20 amino acids next to the transmembrane domain, which might alter its functionality. Our findings suggest that this LRR-RLK-ULP might be responsible for FOB resistance in sweet basil and demonstrate the usefulness of the recently sequenced basil genome for QTL mapping and gene mining.

Characterization of natural antisense transcripts arisen from the locus encoding Toxoplasma gondii ubiquitin-like protease.

Natural antisense transcripts (NATs) are non-protein coding RNAs that could play an important role in regulating the expression of their counterpart protein encoding sense transcript. Although NATs are widespread in most eukaryotic genomes, very little is known about their functions. This study focuses on gaining a better understanding of the function of NATs in Toxoplasma gondii, a pathogenic unicellular eukaryote. Previously, we characterized the gene encoding the first committed enzyme in sumoylation, named ubiquitin-like protease 1 (TgUlp1), and showed that the expression of TgUlp1 is vital to the life cycle of T. gondii. Interestingly, the locus of TgUlp1 also transcribes a NAT species. Using a dual luciferase assay, we identified the promoter of TgUlp1 NAT to be located within the 3'-region of its counterpart coding sequence. While TgUlp1 mRNA level was detected at a lower level throughout the life cycle of T. gondii, its NAT level was upregulated when the parasite converts from actively replicating tachyzoite form to slowly growing bradyzoite form. To investigate the effect of TgUlp1 NAT on the expression of its counterpart mRNA, we used a reporter system bearing TgUlp1 mRNA sequences and showed that the single-stranded TgUlp1 NAT and its in vitro RNase III processed products have the ability to lower the expression of the reporter system. Using a transgenic Dicer-knockout (TgDicer-KO) strain, we showed that TgDicer is required for the function of TgUlp1 NAT in vivo. The findings strongly suggest that the RNA interference pathway is necessary for the function of TgUlp1 NAT.

A novel approach for production of an active N-terminally truncated Ulp1 (SUMO protease 1) catalytic domain from Escherichia coli inclusion bodies.

The SUMO fusion system is widely used to facilitate recombinant expression and production of difficult-to-express proteins. After purification of the recombinant fusion protein, removal of the SUMO-tag is accomplished by the yeast cysteine protease, SUMO protease 1 (Ulp1), which specifically recognizes the tertiary fold of the SUMO domain. At present, the expression of the catalytic domain, residues 403-621, is used for obtaining soluble and biologically active Ulp1. However, we have observed that the soluble and catalytically active Ulp1403-621 inhibits the growth of E. coli host cells. In the current study, we demonstrate an alternative route for producing active Ulp1 catalytic domain from a His-tagged N-terminally truncated variant, residues 416-621, which is expressed in E. coli inclusion bodies and subsequently refolded. Expressing the insoluble Ulp1416-621 variant is advantageous for achieving higher production yields. Approximately 285 mg of recombinant Ulp1416-621 was recovered from inclusion bodies isolated from 1 L of high cell-density E. coli batch fermentation culture. After Ni2+-affinity purification of inactive and denatured Ulp1416-621 in 7.5 M urea, different refolding conditions with varying l-arginine concentration, pH, and temperature were tested. We have successfully refolded the enzyme in 0.25 M l-arginine and 0.5 M Tris-HCl (pH 7) at room temperature. Approximately 80 mg of active Ulp1416-621 catalytic domain can be produced from 1 L of high cell-density E. coli culture. We discuss the applicability of inclusion body-directed expression and considerations for obtaining high expression yields and efficient refolding conditions to reconstitute the active protein fold.

Bioprocess optimization for the overproduction of catalytic domain of ubiquitin-like protease 1 (Ulp1) from S. cerevisiae in E. coli fed-batch culture.

The exploitation of SUMO (small ubiquitin-related modifier) fusion technology at a large scale for the production of therapeutic proteins with an authentic N-terminus is majorly limited due to the higher cost of ScUlp1 protease. Therefore, the cost-effective production of Saccharomyces cerevisiae Ulp1 protease catalytic domain (402-621 aa) was targeted via its cloning under strong T7 promoter with and without histidine tag. The optimization of cultivation conditions at shake flask resulted in ScUlp1 expression of 195 mg/L in TB medium with a specific product yield of 98 mg/g DCW. The leaky expression of the ScUlp1 protease was controlled using the chemically defined minimal medium. The Ni-NTA affinity purification of ScUlp1 was done near homogeneity using different additives (0.1% Triton X-100, 0.01 mM DTT, 0.02 mM EDTA and 1% glycerol) where a product purity of ∼95% with a recovery yield of 80% was obtained. The specific activity of purified ScUlp1 was found to be 3.986 × 105 U/mg. The ScUlp1 protease successfully cleaved the SUMO tag even at 1:10,000 enzyme to substrate ratio with high efficacy and also showed a comparable catalytic efficiency as of commercial control. Moreover, the in vivo cleavage of SUMO tag via co-expression strategy also resulted in more than 80% cleavage of SUMO fusion protein. The optimization of high cell density cultivation strategies and maintenance of higher plasmid stability at bioreactor level resulted in the ScUlp1 production of 3.25 g/L with a specific product yield of 45.41 mg/g DCW when cells were induced at an OD600 of 132 (63.66 g/L DCW).

Toxoplasma ubiquitin-like protease 1, a key enzyme in sumoylation and desumoylation pathways, is under the control of non-coding RNAs.

Sumoylation and desumoylation are reversible pathways responsible for modification of protein structures and functions by the reversible covalent attachment of a small ubiquitin-like modifier (SUMO) peptide. These pathways are important for a wide range of cellular processes and require a steady supply of SUMO, which is generated by an enzymatic reaction catalysed by the ubiquitin-like protease (Ulp) family. Here we show by functional complementation analysis that the Ulp1 of Toxoplasma gondii (TgUlp1) can rescue a growth-deficient phenotype of a yeast-Ulp1 knockout. Recombinant TgUlp1 is an active enzyme capable of removing SUMO from a sumoylated substrate. Using a clonal transgenic strain of T. gondii expressing an epitope-tagged version of TgUlp1, we detected that the expression of TgUlp1 is modulated by Tg-miR-60, the most abundant species of micro RNA found in the T. gondii type 1 strain. The introduction of Tg-miR-60 inhibitor caused an increase in TgUlp1 expression and its enzymatic activity, as well as affecting the parasite's growth fitness. Moreover, we discovered a polyadenylated antisense RNA transcribed from the TgUlp1 locus, referred to as TgUlp1-NAT1 (TgUlp1-natural antisense transcript 1). Both Tg-miR-60 and TgUlp1-NAT1 confer a regulatory function by down-regulating the expression of TgUlp1 and affecting the sumoylation and desumoylation pathways in T. gondii.

A Novel Strategy for the Preparation of Codon-Optimized Truncated Ulp1 and its Simplified Application to Cleavage the SUMO Fusion Protein.

Ubiquitin-like protease 1 (Ulp1) of Saccharomyces cerevisiae emerges as a fundamental tool to obtain the natural N-terminal target protein by cleavage of the small ubiquitin-related modifier (SUMO) fusion protein. However, the costly commercial Ulp1 and its complicated procedures limit its application in the preparation of the target protein with natural N-terminal sequence. Here, we describe the preparation of bioactive codon-optimized recombinant truncated Ulp1 (Leu403-Lys621) (rtUlp1) of S. cerevisiae in Escherichia coli using only one-step with Ni-NTA affinity chromatograph, and the application of rtUlp1 to cleave the SUMO fusion protein by simply mixing the purified rtUlp1, SUMO fusion protein and DL-Dithiothreitol in Tris-HCl buffer. The optimal expression level of non-fusion protein rtUlp1 accounts for approximately 50 % of the total cellular protein and 36% of the soluble form by addition of isopropyl ß-D-l-thiogalactopyranoside at a final concentration of 0.4 mM at 18 °C for 20 h. The purification of target protein rtUlp1 was conducted by Ni-NTA affinity chromatography. The final yield of rtUlp1 was 45 mg/l in flask fermentation with a purity up to 95%. Furthermore, the high purity of rtUlp1 could effectively cleave the SUMO-tTßRII fusion protein (SUMO gene fused to truncated transforming growth factor-beta receptor type II gene) with the above simplified approach, and the specific activity of the rtUlp1 reached up to 2.8 × 10(4) U/mg, which is comparable to the commercial Ulp1. The preparation and application strategy of the rtUlp1 with commonly available laboratory resources in this study will be convenient to the cleavage of the SUMO fusion protein to obtain the natural N-terminal target protein, which can be implemented in difficult-to-express protein functional analysis.