SUMO targeting of a stress-tolerant Ulp1 SUMO protease

SUMO proteases of the SENP/Ulp family are master regulators of both sumoylation and desumoylation and regulate SUMO homeostasis in eukaryotic cells. SUMO conjugates rapidly increase in response to cellular stress, including nutrient starvation, hypoxia, osmotic stress, DNA damage, heat shock, and other proteotoxic stressors. Nevertheless, little is known about the regulation and targeting of SUMO proteases during stress. To this end we have undertaken a detailed comparison of the SUMO-binding activity of the budding yeast protein Ulp1 (ScUlp1) and its ortholog in the thermotolerant yeast Kluyveromyces marxianus, KmUlp1. We find that the catalytic UD domains of both ScUlp1 and KmUlp1 show a high degree of sequence conservation, complement a ulp1 Delta mutant in vivo, and process a SUMO precursor in vitro. Next, to compare the SUMO-trapping features of both SUMO proteases we produced catalytically inactive recombinant fragments of the UD domains of ScUlp1 and KmUlp1, termed ScUTAG and KmUTAG respectively. Both ScUTAG and KmUTAG were able to efficiently bind a variety of purified SUMO isoforms and bound immobilized SUMO1 with nanomolar affinity. However, KmUTAG showed a greatly enhanced ability to bind SUMO and SUMO-modified proteins in the presence of oxidative, temperature and other stressors that induce protein misfolding. We also investigated whether a SUMO-interacting motif (SIM) in the UD domain of KmULP1 that is not conserved in ScUlp1 may contribute to the SUMO-binding properties of KmUTAG. In summary, our data reveal important details about how SUMO proteases target and bind their sumoylated substrates, especially under stress conditions. We also show that the robust pan-SUMO binding features of KmUTAG can be exploited to detect and study SUMO-modified proteins in cell culture systems

Omics-based molecular techniques in oral pathology centred cancer: Prospect and challenges in Africa

: The completion of the human genome project and the accomplished milestones in the human proteome project; as well as the progress made so far in computational bioinformatics and “big data” processing have contributed immensely to individualized/personalized medicine in the developed world.At the dawn of precision medicine, various omics-based therapies and bioengineering can now be applied accurately for the diagnosis, prognosis, treatment, and risk stratifcation of cancer in a manner that was hitherto not thought possible. The widespread introduction of genomics and other omics-based approaches into the postgraduate training curriculum of diverse medical and dental specialties, including pathology has improved the profciency of practitioners in the use of novel molecular signatures in patient management. In addition, intricate details about disease disparity among diferent human populations are beginning to emerge. This would facilitate the use of tailor-made novel theranostic methods based on emerging molecular evidences

KmUTAG SUMO-binding under stress conditions.

<p>(<b>A</b>) Analysis of SUMO-binding under heat stress. Recombinant KmUTAG or ScUTAGs were incubated with SUMO1 beads in the presence or absence of the indicated treatments: with TCEP at room temperature (lanes 2 and 3) and at 42°C (lanes 4 and 5). Subsequently, SUMO beads with bound UTAG proteins were visualized and quantitated using a BioRad imager and BioRad Image Lab software. (<b>B</b>) Three recombinant UTAG proteins, ScUTAG, ScUTAG containing the putative KmSIM and KmUTAG (lanes 2–4), were incubated with SUMO1-conjugated agarose beads in the presence of a reducing agent TCEP [5mM] or 1% hydrogen peroxide (H₂O₂). SUMO1-bound UTAG proteins in TCEP-containing buffer (lanes 5–7) or in peroxide-containing buffer (lanes 8–10) were eluted and visualized as above. Reduced binding of both ScUTAG proteins to SUMO1 beads was quantitated as above. (<b>C</b>) Analysis of SUMO-binding in the presence of various concentrations of hydrogen peroxide (H₂O₂). KmUTAG or ScUTAG proteins were incubated with SUMO1 beads in the presence or absence of the indicated concentrations of hydrogen peroxide (lanes 4–9), without hydrogen peroxide (lanes 2 and 3). Subsequently, eluted UTAG proteins were visualized and quantitated as above. (<b>D & E</b>) Analysis of SUMO-binding in the presence of various concentrations of urea and SDS. Recombinant KmUTAG or ScUTAGs in the presence of urea (0.02 M, 0.2 M, 2.0 M) or SDS (0.02%, 0.2%, 2.0%) as indicated. All binding reactions were performed in the presence of 5mM TCEP. SUMO1 binding reactions without urea and SDS are included as controls (<b>F</b>) Binding of UTAG to a SUMO-fusion protein. Recombinant HIS6-SUMO-CAT (a linear fusion protein of a HIS6 affinity tag, Smt3/SUMO, and chloramphenicol acetyl transferase) was incubated with KmUTAG or ScUTAG (these UTAGs were produced as fusions with the maltose binding protein MBP). Individual reactions were incubated as follows: at 42°C (lanes 2 and 3), in the presence of 0.6% hydrogen peroxide (H₂O₂) (lanes 4 and 5), or in the presence of 5mM TCEP (lanes 6 and 7). After incubation at the stated conditions, amylose beads were used to pull down the UTAG and associated SUMO-CAT. UTAG and co-purifying proteins were eluted with 2x SDS-PAGE sample buffer, visualized and quantitated as detailed above. Also shown for comparison and as controls are samples of recombinant SUMO-CAT (lane 8) and SUMO1 purified KmUTAG (lane 9) and ScUTAG (lane 10). Graphic representation to the right of A-F indicate which proteins were linked to SUMO beads or amylose resin and which proteins were pulled down.</p

UTAG expression in mammalian cells.

<p>(<b>A</b>) UTAG localization to sites of sumoylation in mammalian cells. mCherry-tagged KmUTAG and pEYFP-SUMO1 were transfected into mammalian cells that were grown on cover slips. Cells were then fixed, stained with DAPI, and visualized using a confocal microscope. An overlay of DAPI, mCherry, and YFP images is shown in the lower right corner (merge). Expression of kmUTAG did not alter the distribution or localization of YFP-SUMO (<b>B</b>) Using UTAG to pulldown SUMO-modified proteins from mammalian cell extracts. Whole cell extracts (WCE) of 786–0 renal carcinoma cells transfected with YFP-SUMO1, control GFP or untransfected (none). Pulldowns using recombinant KmUTAG on the right. Note high molecular weight YFP-SUMO1 conjugates in the pulldown of YFP-SUMO1. The graphic representation to the right indicates that KmUTAG was immobilized on amylose beads (circle) and that YFP-SUMO conjugates were pulled down. (C) 5ug KmUTAG, SCUTAG or no protein (mock) were added to 100ul diluted cell extracts (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191391#sec010" target="_blank">Materials & methods</a>) and nutated in the presence of nickel beads for 1 hour at 25°C. Eluted proteins were run on SDS-PAGE, western blotted with anti SUMO1 antibody, and quantitated using a c-DIGIT scanner (Li-Cor Biosciences). 20% of input whole cell extract (WCE) probed with the anti SUMO1 antibody is shown for comparison.</p

Analysis of binding kinetics of the KmUTAG protein to SUMO.

<p>(<b>A</b>) Lanes 2–7: Binding of KmUTAG to SUMO1 beads over time (min) or in the presence or absence of hydrogen peroxide (H₂O₂) as indicated. Lane 2: total protein in each binding reaction. (<b>B</b>) Retention of bound KmUTAG (Km) or ScUTAG (Sc) on SUMO2 beads over time (min). (<b>C</b>) Sensorgrams for KmUTAG (top) and ScUTAG (bottom) binding to biotinylated SUMO1. Analyte UTAG concentrations were 250 nM (red), 125 nM (orange), 63 nM (yellow), 31 nM (green), 16 nM (blue), 8 nM (purple) and 4 nM (magenta). Raw data (points) are shown with fits (solid lines) to a global one-state binding model. Association was 0–300 s. Dissociation was 300–600 s. Graphic representations to the right of A and B indicate that SUMO1 or 2 was immobilized on the beads (circles) and that KmUTAG was precipitated.</p

KmUTAG is a SUMO-binding protein.

<p>(<b>A</b>) Two-hybrid assay demonstrating that catalytically inactive KmUTAG, ScUTAG, and ScUTAG^SIM prey constructs (in <i>LEU2</i> marked pOAD) interact with a BD-SMT3/SUMO bait construct (in <i>TRP1</i> marked pOBD2) and activate a <i>HIS3</i> reporter construct as assayed on –Trp-Leu-His media. All transformed strains grow on –Trp -Leu media. Catalytically active KmUD and ScUD fail to activate the reporter. (<b>B</b>) Binding of recombinant KmUD, ScUTAG and KmUTAG to SUMO1, SUMO2, and SUMO3-conjugated agarose beads. Proteins were bound to the indicated beads in the presence of the reducing agent TCEP in SUMO Protease Buffer (SPB: See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191391#sec010" target="_blank">Materials and methods</a>). After 3 washes proteins were eluted, resolved on SDS-PAGE gels, and stained using a Coomassie G-250 stain. Lane 1 in all figures corresponds to the protein ladder with molecular weights indicated in kDa. (<b>C</b>) Binding of recombinant MBP (maltose binding protein) fusions of KmUD, ScUTAG and KmUTAG to a soluble SUMO fusion protein (SUMO-CAT), SUMO1, and pro-SUMO. After incubation in SPB with TCEP, protein complexes were pulled down using amylose resin. Proteins were resolved on SDS-PAGE gels, and stained as above. Arrows indicate UD and UTAG proteins. Also indicated are SUMO-CAT and SUMO1. 6μg of each protein was used for binding assay and half of the binding reactions were run on the gel (compare also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191391#pone.0191391.g002" target="_blank">Fig 2B</a>). (*) indicates 1μg of SUMO-CAT that was included as a loading control in the lane with the ladder. SUMO1 and proSUMO1 inputs are show in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191391#pone.0191391.s002" target="_blank">S2 Fig</a> (<b>D</b>) KmUTAG binds an <i>in vitro</i>–sumoylated fragment of RanGAP1 (RG1^f). RG1^f (lane 2) was sumoylated <i>in vitro</i> with SUMO1 to produce RG1^f –SUMO1 (lane 1) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191391#sec010" target="_blank">Materials and methods</a>). RG1^f –SUMO1 was incubated with KmUTAG or KmUD as indicated and pulled down using amylose resin. Proteins were resolved on SDS-PAGE gels and western blotted with an antibody to SUMO1. In this western blot the anti SUMO1 antibody cross-reacted with KmUTAG, KmUD and RG1^f as indicated on the blot (*). Note that RG1^f –SUMO1 was only pulled down with KmUTAG (lane 3), but not KmUD (lane 4). Sumoylated RG1 is cleaved by catalytically active KmUD1 (lane 4) and is then removed with the washes before elution (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191391#sec010" target="_blank">Materials and methods</a>). Graphic representations to the right of B,C,D indicate whether SUMO or a protease domain was immobilized on the beads (circles) and which protein was bound.</p

Alignment and features of the Ulp1 SUMO protease from <i>Saccaromyces cerevisiae</i> and <i>Kluyveromyces marxianus</i>.

<p>(<b>A)</b> Alignment of Ulp1 from <i>Kluyveromyces marxianus</i> (top) and <i>Saccharomyces cerevisiae</i> (bottom). The arrow indicates the catalytic cysteine at position C580 in ScUlp1 and C517 in kmUlp1. The yellow line indicates the region of the conserved catalytic UD domain of Ulp1 (NCBI #COG5160) characterized in this study and the blue highlight indicates a previously described SUMO-binding surface. A potential SIM (VDILD) that is present in KmULP1 but not ScULP1 is marked with a red box. (<b>B)</b> Schematic representation of SUMO proteases, truncations, and mutants studied in this work. (<b>C</b>) Three dimensional representation of the co-crystal structure of the catalytic domain of Ulp1 (Ulp1-UD, magenta) with yeast small ubiquitin-like modifier (SUMO/Smt3, blue). Indicated in yellow are the residues that correspond to the predicted SIM domain (VDILD) present in KmUlp1. The model was derived using MMDB database entry 13315.</p