ULP-2 SUMO protease regulates UPRmt and mitochondrial homeostasis in Caenorhabditis elegans.

Mitochondria are the powerhouses of cells, responsible for energy production and regulation of cellular homeostasis. When mitochondrial function is impaired, a stress response termed mitochondrial unfolded protein response (UPRmt) is initiated to restore mitochondrial function. Since mitochondria and UPRmt are implicated in many diseases, it is important to understand UPRmt regulation. In this study, we show that the SUMO protease ULP-2 has a key role in regulating mitochondrial function and UPRmt. Specifically, down-regulation of ulp-2 suppresses UPRmt and reduces mitochondrial membrane potential without significantly affecting cellular ROS. Mitochondrial networks are expanded in ulp-2 null mutants with larger mitochondrial area and increased branching. Moreover, the amount of mitochondrial DNA is increased in ulp-2 mutants. Downregulation of ULP-2 also leads to alterations in expression levels of mitochondrial genes involved in protein import and mtDNA replication, however, mitophagy remains unaltered. In summary, this study demonstrates that ULP-2 is required for mitochondrial homeostasis and the UPRmt.

1,10-phenanthroline inhibits sumoylation and reveals that yeast SUMO modifications are highly transient.

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.

Calcium-assisted sortase A cleavage of SUMOylated metallothionein constructs leads to high-yield production of human MT3.

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).

Robust production of active Ulp1 (SUMO protease) from inclusion bodies.

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.

Point mutations of homologs as an adaptive solution to the gene loss.

Gene loss is common and influences genome evolution trajectories. Multiple adaptive strategies to compensate for gene loss have been observed, including copy number gain of paralogous genes and mutations in genes of the same pathway. By using the Ubl-specific protease 2 (ULP2) eviction model, we identify compensatory mutations in the homologous gene ULP1 by laboratory evolution and find that these mutations are capable of rescuing defects caused by the loss of ULP2. Furthermore, bioinformatics analysis of genomes of yeast gene knockout library and natural yeast isolate datasets suggests that point mutations of a homologous gene might be an additional mechanism to compensate for gene loss.

Sumoylation is Largely Dispensable for Normal Growth but Facilitates Heat Tolerance in Yeast.

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 (∼75% 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 ∼5% 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.

A nuclear pore complex-associated regulation of SUMOylation in meiosis.

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.

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.

Effective Strategies for Heterologous Expression of Plant Heterotrimeric G-protein γ Subunits without Gß Subunit Partners.

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.

Liquid biopsy detection of genomic alterations in pediatric brain tumors from cell-free DNA in peripheral blood, CSF, and urine.

BACKGROUND: The ability to identify genetic alterations in cancers is essential for precision medicine; however, surgical approaches to obtain brain tumor tissue are invasive. Profiling circulating tumor DNA (ctDNA) in liquid biopsies has emerged as a promising approach to avoid invasive procedures. Here, we systematically evaluated the feasibility of profiling pediatric brain tumors using ctDNA obtained from plasma, cerebrospinal fluid (CSF), and urine. METHODS: We prospectively collected 564 specimens (257 blood, 240 urine, and 67 CSF samples) from 258 patients across all histopathologies. We performed ultra-low-pass whole-genome sequencing (ULP-WGS) to assess copy number variations and estimate tumor fraction and developed a pediatric CNS tumor hybrid capture panel for deep sequencing of specific mutations and fusions. RESULTS: ULP-WGS detected copy number alterations in 9/46 (20%) CSF, 3/230 (1.3%) plasma, and 0/153 urine samples. Sequencing detected alterations in 3/10 (30%) CSF, 2/74 (2.7%) plasma, and 0/2 urine samples. The only positive results were in high-grade tumors. However, most samples had insufficient somatic mutations (median 1, range 0-39) discoverable by the sequencing panel to provide sufficient power to detect tumor fractions of greater than 0.1%. CONCLUSIONS: Children with brain tumors harbor very low levels of ctDNA in blood, CSF, and urine, with CSF having the most DNA detectable. Molecular profiling is feasible in a small subset of high-grade tumors. The level of clonal aberrations per genome is low in most of the tumors, posing a challenge for detection using whole-genome or even targeted sequencing methods. Substantial challenges therefore remain to genetically characterize pediatric brain tumors from liquid biopsies.

Cell-Based Assays for Smoothened Ubiquitination and Sumoylation.

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.

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

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.

SMC complexes are guarded by the SUMO protease Ulp2 against SUMO-chain-mediated turnover.

Structural maintenance of chromosomes (SMCs) complexes, cohesin, condensin, and Smc5/6, are essential for viability and participate in multiple processes, including sister chromatid cohesion, chromosome condensation, and DNA repair. Here we show that SUMO chains target all three SMC complexes and are antagonized by the SUMO protease Ulp2 to prevent their turnover. We uncover that the essential role of the cohesin-associated subunit Pds5 is to counteract SUMO chains jointly with Ulp2. Importantly, fusion of Ulp2 to kleisin Scc1 supports viability of PDS5 null cells and protects cohesin from proteasomal degradation mediated by the SUMO-targeted ubiquitin ligase Slx5/Slx8. The lethality of PDS5-deleted cells can also be bypassed by simultaneous loss of the proliferating cell nuclear antigen (PCNA) unloader, Elg1, and the cohesin releaser, Wpl1, but only when Ulp2 is functional. Condensin and Smc5/6 complex are similarly guarded by Ulp2 against unscheduled SUMO chain assembly, which we propose to time the availability of SMC complexes on chromatin.

Case Report: Circulating Tumor DNA Fraction Analysis Using Ultra-Low-Pass Whole-Genome Sequencing Correlates Response to Chemoradiation and Recurrence in Stage IV Small-Cell Carcinoma of the Cervix - A Longitudinal Study.

Neuroendocrine carcinoma of the cervix is a rare and aggressive form of cervical cancer that presents with frequent metastasis at diagnosis and high recurrence rates. Primary treatment is multimodal, which often includes chemotherapy with or without radiation therapy. There are no data available to guide treatment for recurrence, and second-line therapies are extrapolated from small-cell lung carcinoma data. Close monitoring of these patients for recurrence is paramount. Evaluation of circulating tumor DNA (ctDNA) in the peripheral blood is an attractive approach due to its non-invasive nature. Ultra-low-pass whole-genome sequencing (ULP-WGS) can assess tumor burden and response to therapy and predict recurrence; however, data are lacking regarding the role of ULP-WGS in small-cell carcinoma of the cervix. This study demonstrates a patient whose response to chemotherapy and cancer recurrence was accurately monitored by ctDNA analysis using ULP-WGS and confirmed with radiologic imaging findings.

Numerical Modelling Assisted Design of a Compact Ultrafiltration (UF) Flat Sheet Membrane Module.

The increasing adoption of ultra-low pressure (ULP) membrane systems for drinking water treatment in small rural communities is currently hindered by a limited number of studies on module design. Detailed knowledge on both intrinsic membrane transport properties and fluid hydrodynamics within the module is essential in understanding ULP performance prediction, mass transfer analysis for scaling-up between lab-scale and industrial scale research. In comparison to hollow fiber membranes, flat sheet membranes present certain advantages such as simple manufacture, sheet replacement for cleaning, moderate packing density and low to moderate energy usage. In the present case study, a numerical model using computational fluid dynamics (CFD) of a novel custom flat sheet membrane module has been designed in 3D to predict fluid flow conditions. The permeate flux through the membrane decreased with an increase in spacer curviness from 2.81 L/m2h for no (0%) curviness to 2.73 L/m2h for full (100%) curviness. A parametric analysis on configuration variables was carried out to determine the optimum design variables and no significant influence of spacer inflow or outflow thickness on the fluid flow were observed. The numerical model provides the necessary information on the role of geometrical and operating parameters for fabricating a module prototype where access to technical expertise is limited.

A 6-Transistor Ultra-Low Power CMOS Voltage Reference with 0.02%/V Line Sensitivity.

This work presents a technique for design of ultra-low power (ULP) CMOS voltage references achieving extremely low line sensitivity while maintaining state-of-the-art temperature insensitivity through the use of a 6-transistor (6T) structure. The proposed technique demonstrates good performance in sub-100 nm CMOS technologies. The 65-nm CMOS implementation occupies only 840 µm2 of area and consumes 28.6 pA from a 0.5 V supply. Measurements from 6 samples from the same wafer show an average line sensitivity of 0.02 %/V, a 10X improvement over previous 65 nm implementations, and an average temperature coefficient of 99.2 ppm/°C.

The Leishmania donovani SENP Protease Is Required for SUMO Processing but Not for Viability.

The protozoan parasite Leishmania donovani is part of an early eukaryotic branch and depends on post-transcriptional mechanisms for gene expression regulation. This includes post-transcriptional protein modifications, such as protein phosphorylation. The presence of genes for protein SUMOylation, i.e., the covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides, in the Leishmania genomes prompted us to investigate the importance of the sentrin-specific protease (SENP) and its putative client, SUMO, for the vitality and infectivity of Leishmania donovani. While SENP null mutants are viable with reduced vitality, viable SUMO null mutant lines could not be obtained. SUMO C-terminal processing is disrupted in SENP null mutants, preventing SUMO from covalent attachment to proteins and nuclear translocation. Infectivity in vitro is not affected by the loss of SENP-dependent SUMO processing. We conclude that SENP is required for SUMO processing, but that functions of unprocessed SUMO are critical for Leishmania viability.

Unsupported liquid-state platform for SERS-based determination of triazophos.

A highly reproducible surface-enhanced Raman scattering (SERS) unsupported liquid-state platform (ULP) was developed for accurate quantitative determination of triazophos. Herein, citrate-reduced Ag NPs suspension was concentrated and placed in a stainless steel perforated template to form the SERS ULP. The relative standard deviation of the SERS measurements was less than 5% (n ≥ 10), and the R2 of the calibration curve was 0.994. The developed SERS ULP was applied for determination of triazophos in spiked agricultural products (rice, cabbage, and apple). Experiment results showed that the coefficient of variation ranged from 5.3 to 6.2% for intra-day and from 5.5 to 6.3% for inter-day (n = 3), which proved excellent SERS reproducibility. Moreover, the results were in good agreement with those from HPLC analysis. As a liquid-state SERS substrate, the highly reproducible ULP can perform precision quantitative analysis without surface modification of NPs, which is a significant improvement. This method provides a new perspective for quantitative SERS analysis of pesticide residues. Graphical abstract.

Expression and Purification of an Intrinsically Disordered Protein.

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.

A simple and rapid protein purification method based on cell-surface display of SUMO-fused recombinant protein and Ulp1 protease.

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.