SUMO-modified Proteins Research Articles

SUMO-1 plays crucial roles for spindle organization, chromosome congression, and chromosome segregation during mouse oocyte meiotic maturation.

Small ubiquitin-related modifier-1 (SUMO-1)-dependent modifications of many target proteins are involved in a range of intracellular processes. Previous studies reported the localization of SUMO-1 during oocyte meiosis, and that overexpression of Sentrin/SUMO-specific protease 2 (SENP2), a de-SUMOylation protease, altered SUMO-modified proteins, and caused defects in metaphase-II spindle organization. In this study, we detailed the consequences of SUMO-1-mediated SUMOylation by either inhibition of SUMO-1 or UBC9 with a specific antibody or their depletion by specific siRNA microinjection. Inhibition or depletion of SUMO-1 or UBC9 in germinal vesicle (GV)-stage oocytes decreased the rates of germinal vesicle breakdown and first polar body (PB1) extrusion; caused defective spindle organization and misaligned chromosomes; and led to aneuploidy in matured oocytes. Stage-specific antibody injections suggested that SUMO-1 functions before anaphase I during PB1 extrusion. Further experiments indicated that the localization of γ-tubulin was disordered after SUMO-1 inhibition, and that SUMO-1 depletion disrupted kinetochore-microtubule attachment at metaphase I. Moreover, SUMO-1 inhibition resulted in less-condensed chromosomes, altered localization of REC8 and securin, and reduced BUBR1 accumulation at the centromere. On the other hand, overexpression of SUMO-1 in GV-stage oocytes had no significant effect on oocyte maturation. In conclusion, our results implied that SUMO-1 plays crucial roles during oocyte meiotic maturation, specifically involving spindle assembly and chromosome behavior, by regulating kinetochore-microtubule attachment and the localization of γ-tubulin, BUBR1, REC8, and securin.

Important role of SUMOylation of Spliceosome factors in prostate cancer cells.

Sentrin/SUMO (small ubiquitin-like modifier)-specific proteases (SENPs) have been implicated in the development of prostate cancer. However, due to the low abundance of SUMO-modified proteins and high activity of SENPs, the SUMO substrates affected by SENPs in prostate cancer cells are largely unknown. Here, we identified SI2, a novel cell-permeable SENP-specific inhibitor, by high-throughput screening. Using SI2 as a way of inhibiting the activity of SENPs and the SUMO stably transfected PC3 cells as a prostate cancer model, in combination with the stable isotope labeling with amino acids (SILAC) quantitative proteomic technique, we identified more than 900 putative target proteins of SUMO, in which 231 proteins were further subjected to bioinformatic analysis. In the highly enriched spliceosome pathway, we validated that USP39, HSPA1A, and HSPA2 were novel target proteins of SUMO. Furthermore, we demonstrated that K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. Mutation of these SUMO modification sites of USP39 further promoted the proliferation-enhancing effect of USP39 on prostate cancer cells. This study provides the SUMOproteome of PC3 cells and reveals that SUMOylation of spliceosome factors may be implicated in the pathogenesis of prostate cancer. Optimization of SI2 for isotype-specific SENP inhibitors warrants further investigation.

Versatile recombinant SUMOylation system for the production of SUMO-modified protein.

Posttranslational modification by small ubiquitin-like modifiers (SUMO) is being associated with a growing number of regulatory functions in diverse cellular processes. The biochemical investigation into the underlying molecular mechanisms, however, has been lagging behind due to the difficulty to generate sufficient amounts of recombinant SUMOylated proteins. Here, we present two newly designed two-component vector systems for the expression and purification of SUMO-modified target proteins in Escherichia coli. One system consists of a vector for SUMO conjugation, expressing human SUMO-activating (SAE1/SAE2) and conjugating (Ubc9) enzymes together with His6-tagged SUMO1, 2 or 3, that can be combined with commonly used expression constructs for any gene of interest. To facilitate SUMOylation of targets normally requiring a SUMO-E3 ligase for efficient modification, a second system is designed to express the target protein as a fusion with the human SUMO-conjugating enzyme Ubc9, thus compensating the absence of a potential SUMO ligase. We demonstrate the proficiency of these systems by SUMOylation of two DNA repair proteins, the thymine DNA glycosylase (TDG) and XRCC1, and describe purification schemes for SUMOylated proteins in native and active form. This SUMO toolbox facilitates “in-cell” and “in-extract” production and purification of recombinant SUMO-modified target proteins for functional and structural analysis.

Spatiotemporal distribution of SUMOylation components during mouse brain development

Posttranslational modification of proteins might play an important role in brain cellular dynamics via the rapid turnover or functional change of critical proteins controlling neuronal differentiation or synaptic transmission. Small ubiquitin-like modifier protein (SUMO) is a family of ubiquitin-like small proteins that are covalently attached to target proteins to modify their function posttranslationally. Many cellular processes, such as transcription and protein trafficking, are regulated by SUMOylation, but its functional significance in the brain remains unclear. Although developmental regulation of SUMOylation levels in rat brain was recently demonstrated, no comparative immunohistochemical analysis of the cellular distribution profiles of SUMOylation components, including SUMO1, SUMO2/3, and Ubc9, has been undertaken so far. The present study used immunohistochemical and immunoblot analysis with the different developmental stages of mice and demonstrated the developmentally regulated distribution of SUMO1, SUMO2/3, and Ubc9 in the brain. During embryonic development, SUMOylation by SUMO1 and SUMO2/3 occurred in the nucleoplasm of nestin-positive neural stem cells. Although the total amount of SUMO-modified proteins decreased during postnatal brain development, intense and persistent accumulation of SUMO2/3 was detected throughout life in neural progenitor populations in neurogenic regions, including the subventricular zone and the hippocampal subgranular zone. In contrast, many neurons in the adult brain accumulated SUMO1 rather than SUMO2/3. Heavy immunoreactivity of SUMO1 was found in large projection neurons in the brainstem, whereas SUMO2/3 was almost absent from these areas. This heterogeneous distribution implies that both proteins play a specific and unique role in the brain.

RNF 4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

SUMOylation negatively modulates target gene occupancy of the KDM5B, a histone lysine demethylase

The histone lysine demethylase KDM5B plays key roles in gene repression by demethylating trimethylated lysine 4 of histone H3 (H3K4me3), a modification commonly found at the promoter region of actively transcribed genes. KDM5B is known to regulate the expression of genes involved in cell cycle progression; however, little is known about the post-translational modifications that regulate KDM5B. Herein, we report that KDM5B is SUMOylated at lysine residues 242 and 278 and that the ectopic expression of the hPC2 SUMO E3 ligase enhances this SUMOylation. Interestingly, the levels of KDM5B and its SUMOylated forms are regulated during the cell cycle. KDM5B is modulated by RNF4, an E3 ubiquitin ligase that targets SUMO-modified proteins to proteasomal degradation. Digital gene expression analyses showed that cells expressing the SUMOylation-deficient KDM5B harbor repressed mRNA expression profiles of cell cycle and DNA repair genes. Chromatin immunoprecipitations confirmed some of these genes as KDM5B targets, as they displayed reduced H3K4me3 levels in cells ectopically expressing KDM5B. We propose that SUMOylation by hPC2 regulates the activity of KDM5B.

The CacyBP/SIP Protein is Sumoylated in Neuroblastoma NB2a Cells

The Calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein is highly expressed in mammalian brain as well as in neuroblastoma NB2a cells and pheochromocytoma PC12 cells. This protein interacts with several targets such as cytoskeletal proteins or ERK1/2 kinase and seems to be involved in many cellular processes. In this work we examined a post-translational modification of CacyBP/SIP which might have an effect on its function. Since theoretical analysis of the amino acid sequence of CacyBP/SIP indicated several lysine residues which could potentially be sumoylated we checked experimentally whether this protein might be modified by SUMO attachment. We have shown that indeed CacyBP/SIP bound the E2 SUMO ligase, Ubc9, in neuroblastoma NB2a cell extract and was sumoylated in these cells. By fractionation of NB2a cell extract we have found that, contrary to the majority of SUMO-modified proteins, sumoylated CacyBP/SIP is present in the cytoplasmic and not in the nuclear fraction. We have also established that lysine 16 is the residue which undergoes sumoylation in the CacyBP/SIP protein.

Identification of SUMO-modified proteins by affinity purification and tandem mass spectrometry in Arabidopsis thaliana

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in eukaryotes. Although more than 200 SUMO target proteins have been characterized in yeasts and animals, only very few SUMO targets are known in plants. Here, to identify putative SUM1 associating proteins in Arabidopsis we developed a proteomics-type protocol employing a His-GST-AtSUM1 column conjugated to Affi-gel beads with total protein extracts from 3–4 week old seedlings. AtSUM1 binding proteins and/or sumoylated targets were obtained by a change in pH of the eluant and identified by mass spectrometry. Fifteen candidate SUMO-modified proteins have so far been identified. Candidate proteins were analyzed by a split ubiquitination assay in yeast and additionally by split-luciferase complement assays in planta to confirm binding specificity and activity to AtSUM1, and productive sumoylation through in vivo assays in E. coli. Among proteins found reliably sumoylated are AtMYB31 (transcription factor), ACS7 (1-amino-cyclopropane-1-carboxylate synthase 7), and Rho GDI1 (Rho GDPdissociation inhibitor family protein). These proteins will provide a basis for studying functions of SUMOylation in the regulation of diverse processes in Arabidopsis.

Senp1 Is Essential for Desumoylating Sumo1-Modified Proteins but Dispensable for Sumo2 and Sumo3 Deconjugation in the Mouse Embryo

Posttranslational modification with small ubiquitin-like modifier (Sumo) regulates numerous cellular and developmental processes. Sumoylation is dynamic with deconjugation by Sumo-specific proteases (Senps) regulating steady-state levels. Different Senps are found in distinct subcellular domains, which may limit their deconjugation activity to colocalizing Sumo-modified proteins. Invitro, Senps can discriminate between the different Sumo paralogs: Sumo1 versus the highly related Sumo2 and Sumo3 (Sumo2/3), which can form poly-Sumo chains. However, a full understanding of Senp specificity invivo isstill lacking. Here, using biochemical and genetic approaches, we establish that Senp1 has an essential, nonredundant function to desumoylate Sumo1-modified proteins during mouse embryonic development. Senp1 specificity for Sumo1 conjugates represents an intrinsic function and not simply a product of colocalization. In contrast, Senp1 has only a limited role in Sumo2/3 desumoylation, although it may regulate Sumo1-mediated termination of poly-Sumo2/3 chains.

Analysis of SUMOylated proteins using SUMO-traps

SUMO-modified proteins are recognized by SUMO interacting motifs (SIMs), thus triggering diverse cellular responses. Here SIMs were used to develop SUMO-traps to capture endogenous SUMOylated proteins. Our results show that these small peptides are transferable motifs that maintain their SUMO binding capacity when fused to the heterologous carrier protein GST. The tandem disposition of SIMs increases the binding capacity of SUMO-traps to specifically interact with polySUMO but not poly-Ubiquitin chains. We demonstrate that this SUMO capturing system purifies SUMOylated proteins such as IκBα, PTEN, PML or p53 in vitro and in vivo. These properties can be used to explore the many critical functions regulated by protein SUMOylation.

Decoding the SUMO signal

SUMO (small ubiquitin-like modifier) emerged from the shadow of the well-established ubiquitin some 15years ago when it was shown that a distinct conjugation pathway was responsible for SUMO modification. Since then it has been established that SUMO modifies over a thousand substrates and plays diverse roles in many important biological processes. Recognition of SUMO is mediated by short peptide sequences known as SIMs (SUMO-interaction motifs) that allow effector proteins to engage SUMO-modified substrates. Like ubiquitin, SUMO can form polymeric chains, and these chains can be recognized by proteins containing multiple SIMs. One protein that contains such a sequence of SIMs also contains a RING (really interesting new gene) domain that is the hallmark of a ubiquitin E3 ligase. This ubiquitin ligase known as RNF4 (RING finger protein 4) has the unique property that it can recognize SUMO-modified proteins and target them for ubiquitin-mediated proteolysis. Structural and biochemical analyses of RNF4 has shed light on the long sought after mechanism of ubiquitin transfer and illustrates how its RING domain primes the ubiquitin-loaded E2 for catalysis.

A novel approach to the analysis of SUMOylation with the independent use of trypsin and elastase digestion followed by database searching utilising consecutive residue addition to lysine

Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site-directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy. SUMOylated protein samples were digested with a number of enzymes and the resulting peptides separated using liquid chromatography. Analysis was carried out on both linear ion trap Orbitrap and quadrupole-time-of-flight (Q-TOF)-based mass spectrometers equipped with electrospray ionisation. The data files were subsequently searched using the Mascot algorithm with multiple variable tag modifications corresponding to SUMO-derived fragments. The utility of this approach was demonstrated with di-SUMO 2, di-SUMO 3, SUMO 1-RanGap(418-587) 1 and an enriched population of SUMOylated proteins. Unbiased database searches led to the identification of a number of analytically useful isotags ranging in length from two to four residues. Isopeptide fragments were generated including QTGG (di-SUMO-2/3), TGG (di-SUMO-2/3) and GG (SUMO-1). The method was validated by successfully mapping a number of sites of SUMO modification on SUMO-modified proteins enriched from a cell lysate. This combination of relaxed enzyme specificity, shortened isotag generation and unbiased database searching enabled confident identification of novel analytically useful SUMOylated isopeptides without a requirement for mutagenesis.

RNF4 is required for DNA double-strand break repair in vivo

Unrepaired DNA double-strand breaks (DSBs) cause genetic instability that leads to malignant transformation or cell death. Cells respond to DSBs with the ordered recruitment of signaling and repair proteins to the sites of DNA lesions. Coordinated protein SUMOylation and ubiquitylation have crucial roles in regulating the dynamic assembly of protein complexes at these sites. However, how SUMOylation influences protein ubiquitylation at DSBs is poorly understood. We show herein that Rnf4, an E3 ubiquitin ligase that targets SUMO-modified proteins, accumulates in DSB repair foci and is required for both homologous recombination (HR) and non-homologous end joining repair. To establish a link between Rnf4 and the DNA damage response (DDR) in vivo, we generated an Rnf4 allelic series in mice. We show that Rnf4-deficiency causes persistent ionizing radiation-induced DNA damage and signaling, and that Rnf4-deficient cells and mice exhibit increased sensitivity to genotoxic stress. Mechanistically, we show that Rnf4 targets SUMOylated MDC1 and SUMOylated BRCA1, and is required for the loading of Rad51, an enzyme required for HR repair, onto sites of DNA damage. Similarly to inactivating mutations in other key regulators of HR repair, Rnf4 deficiency leads to age-dependent impairment in spermatogenesis. These findings identify Rnf4 as a critical component of the DDR in vivo and support the possibility that Rnf4 controls protein localization at DNA damage sites by integrating SUMOylation and ubiquitylation events.

Herpes Simplex Virus 1 Ubiquitin Ligase ICP0 Interacts with PML Isoform I and Induces Its SUMO-Independent Degradation

Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 localizes to cellular structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10 and disrupts their integrity by inducing the degradation of PML. There are six PML isoforms with different C-terminal regions in ND10, of which PML isoform I (PML.I) is the most abundant. Depletion of all PML isoforms increases the plaque formation efficiency of ICP0-null mutant HSV-1, and reconstitution of expression of PML.I and PML.II partially reverses this improved replication. ICP0 also induces widespread degradation of SUMO-conjugated proteins during HSV-1 infection, and this activity is linked to its ability to counteract cellular intrinsic antiviral resistance. All PML isoforms are highly SUMO modified, and all such modified forms are sensitive to ICP0-mediated degradation. However, in contrast to the situation with the other isoforms, ICP0 also targets PML.I that is not modified by SUMO, and PML in general is degraded more rapidly than the bulk of other SUMO-modified proteins. We report here that ICP0 interacts with PML.I in both yeast two-hybrid and coimmunoprecipitation assays. This interaction is dependent on PML.I isoform-specific sequences and the N-terminal half of ICP0 and is required for SUMO-modification-independent degradation of PML.I by ICP0. Degradation of the other PML isoforms by ICP0 was less efficient in cells specifically depleted of PML.I. Therefore, ICP0 has two distinct mechanisms of targeting PML: one dependent on SUMO modification and the other via SUMO-independent interaction with PML.I. We conclude that the ICP0-PML.I interaction reflects a countermeasure to PML-related antiviral restriction.

Abstract SY07-02: Involvement of ubiquitin ligase RNF4 in the DNA damage response

Abstract SY07-02: Involvement of ubiquitin ligase RNF4 in the DNA damage response

RhoGDI SUMOylation at Lys-138 Increases Its Binding Activity to Rho GTPase and Its Inhibiting Cancer Cell Motility

The Rho GDP dissociation inhibitor (RhoGDI) can bind to small GTPases and keep them in a biologically inactive state in cytoplasm, through which it affects actin polymerization and cell motility. However, mechanisms underlying how RhoGDI regulates Rho GTPase complex formation/membrane extraction/GTPase dissociation remain largely unexplored. Our previous studies reported that X-linked inhibitor of apoptosis protein (XIAP) interacted with RhoGDI via its RING domain and negatively modulated RhoGDI SUMOylation and HCT116 cancer cell migration. Here, we identified that RhoGDI SUMOylation specifically occurred at Lys-138, which was inhibited by XIAP domain. We further demonstrated that RhoGDI SUMOylation at Lys-138 was crucial for inhibiting actin polymerization and cytoskeleton formation as well as cancer cell motility. Moreover, SUMO-RhoGDI had a much higher binding affinity to small Rho GTPase compared with the un-SUMOylated form of RhoGDI. Taken together, our study demonstrated a novel modification of RhoGDI, SUMOylation at Lys-138, which played a key role in regulating Rho GTPase activation in cancer cells. The physiological regulation of RhoGDI SUMOylation by the RING domain of XIAP may account for modulation of cancer cell invasion and metastasis by XIAP.

Sumo-dependent substrate targeting of the SUMO protease Ulp1

BackgroundIn the yeast Saccharomyces cerevisiae, the essential small ubiquitin-like modifier (SUMO) protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood.ResultsUsing a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3)(C580S), that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3)(C580S) to purify Smt3-modified proteins from cell extracts.ConclusionsOur study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates in vivo and in vitro. Furthermore, we found that a substrate-trapping Ulp1(3)(C580S) interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.

A Viral Ubiquitin Ligase Has Substrate Preferential SUMO Targeted Ubiquitin Ligase Activity that Counteracts Intrinsic Antiviral Defence

Intrinsic antiviral resistance represents the first line of intracellular defence against virus infection. During herpes simplex virus type-1 (HSV-1) infection this response can lead to the repression of viral gene expression but is counteracted by the viral ubiquitin ligase ICP0. Here we address the mechanisms by which ICP0 overcomes this antiviral response. We report that ICP0 induces the widespread proteasome-dependent degradation of SUMO-conjugated proteins during infection and has properties related to those of cellular SUMO-targeted ubiquitin ligases (STUbLs). Mutation of putative SUMO interaction motifs within ICP0 not only affects its ability to degrade SUMO conjugates, but also its capacity to stimulate HSV-1 lytic infection and reactivation from quiescence. We demonstrate that in the absence of this viral countermeasure the SUMO conjugation pathway plays an important role in mediating intrinsic antiviral resistance and the repression of HSV-1 infection. Using PML as a model substrate, we found that whilst ICP0 preferentially targets SUMO-modified isoforms of PML for degradation, it also induces the degradation of PML isoform I in a SUMO modification-independent manner. PML was degraded by ICP0 more rapidly than the bulk of SUMO-modified proteins in general, implying that the identity of a SUMO-modified protein, as well as the presence of SUMO modification, is involved in ICP0 targeting. We conclude that ICP0 has dual targeting mechanisms involving both SUMO- and substrate-dependent targeting specificities in order to counteract intrinsic antiviral resistance to HSV-1 infection.

SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin

HP1 enrichment at pericentric heterochromatin is considered important for centromere function. Although HP1 binding to H3K9me3 can explain its accumulation at pericentric heterochromatin, how it is initially targeted there remains unclear. Here, in mouse cells, we reveal the presence of long nuclear noncoding transcripts corresponding to major satellite repeats at the periphery of pericentric heterochromatin. Furthermore, we find that major transcripts in the forward orientation specifically associate with SUMO-modified HP1 proteins. We identified this modification as SUMO-1 and mapped it in the hinge domain of HP1α. Notably, the hinge domain and its SUMOylation proved critical to promote the initial targeting of HP1α to pericentric domains using de novo localization assays, whereas they are dispensable for maintenance of HP1 domains. We propose that SUMO-HP1, through a specific association with major forward transcript, is guided at the pericentric heterochromatin domain to seed further HP1 localization.

Small Ubiquitin-related Modifier (SUMO)-1 Promotes Glycolysis in Hypoxia

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress.