Expression of SUMO and NF-κB genes in hepatitis B virus-associated hepatocellular carcinoma patients: An observational study.

Alterations in signaling pathways and modulation of cell metabolism are associated with the pathogenesis of cancers, including hepatocellular carcinoma (HCC). Small ubiquitin-like modifier (SUMO) proteins and NF-κB family play major roles in various cellular processes. The current study aims to determine the expression profile of SUMO and NF-κB genes in HCC tumors and investigate their association with the clinical outcome of HCC. The expression of 5 genes - SUMO1, SUMO2, SUMO3, NF-κB p65, and NF-κB p50 - was quantified in tumor and adjacent non-tumor tissues of 58 HBV-related HCC patients by real-time quantitative PCR and was analyzed for the possible association with clinical parameters of HCC. The expression of SUMO2 was significantly higher in HCC tumor tissues compared to the adjacent non-tumor tissues (P = .01), while no significant difference in SUMO1, SUMO3, NF-κB p65, and NF-κB p50 expression was observed between HCC tumor and non-tumor tissues (P > .05). In HCC tissues, a strong correlation was observed between the expression of SUMO2 and NF-κB p50, between SUMO3 and NF-κB p50, between SUMO3 and NF-κB p65 (Spearman rho = 0.83; 0.82; 0.772 respectively; P < .001). The expression of SUMO1, SUMO2, SUMO3, NF-κB p65, and NF-κB p50 was decreased in grade 3 compared to grades 1 and 2 in HCC tumors according to the World Health Organization grades system. Our results highlighted that the SUMO2 gene is upregulated in tumor tissues of patients with HCC, and is related to the development of HCC, thus it may be associated with the pathogenesis of HCC.

Broad-spectrum ubiquitin/ubiquitin-like deconjugation activity of the rhizobial effector NopD from Bradyrhizobium (sp. XS1150).

The post-translational modification of proteins by ubiquitin-like modifiers (UbLs), such as SUMO, ubiquitin, and Nedd8, regulates a vast array of cellular processes. Dedicated UbL deconjugating proteases families reverse these modifications. During bacterial infection, effector proteins, including deconjugating proteases, are released to disrupt host cell defenses and promote bacterial survival. NopD, an effector protein from rhizobia involved in legume nodule symbiosis, exhibits deSUMOylation activity and, unexpectedly, also deubiquitination and deNeddylation activities. Here, we present two crystal structures of Bradyrhizobium (sp. XS1150) NopD complexed with either Arabidopsis SUMO2 or ubiquitin at 1.50 Å and 1.94 Å resolution, respectively. Despite their low sequence similarity, SUMO and ubiquitin bind to a similar NopD interface, employing a unique loop insertion in the NopD sequence. In vitro binding and activity assays reveal specific residues that distinguish between deubiquitination and deSUMOylation. These unique multifaceted deconjugating activities against SUMO, ubiquitin, and Nedd8 exemplify an optimized bacterial protease that disrupts distinct UbL post-translational modifications during host cell infection.

A UL26-PIAS1 complex antagonizes anti-viral gene expression during Human Cytomegalovirus infection.

Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon α (IFNA) or tumor necrosis factor α (TNFα). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment similar to ΔUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.

The physical and evolutionary energy landscapes of devolved protein sequences corresponding to pseudogenes.

Protein evolution is guided by structural, functional, and dynamical constraints ensuring organismal viability. Pseudogenes are genomic sequences identified in many eukaryotes that lack translational activity due to sequence degradation and thus over time have undergone "devolution." Previously pseudogenized genes sometimes regain their protein-coding function, suggesting they may still encode robust folding energy landscapes despite multiple mutations. We study both the physical folding landscapes of protein sequences corresponding to human pseudogenes using the Associative Memory, Water Mediated, Structure and Energy Model, and the evolutionary energy landscapes obtained using direct coupling analysis (DCA) on their parent protein families. We found that generally mutations that have occurred in pseudogene sequences have disrupted their native global network of stabilizing residue interactions, making it harder for them to fold if they were translated. In some cases, however, energetic frustration has apparently decreased when the functional constraints were removed. We analyzed this unexpected situation for Cyclophilin A, Profilin-1, and Small Ubiquitin-like Modifier 2 Protein. Our analysis reveals that when such mutations in the pseudogene ultimately stabilize folding, at the same time, they likely alter the pseudogenes' former biological activity, as estimated by DCA. We localize most of these stabilizing mutations generally to normally frustrated regions required for binding to other partners.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

SUMOylated Golgin45 associates with PML-NB to transcriptionally regulate lipid metabolism genes during heat shock stress.

Golgin tethers are known to mediate vesicular transport in the secretory pathway, whereas it is relatively unknown whether they may mediate cellular stress response within the cell. Here, we describe a cellular stress response during heat shock stress via SUMOylation of a Golgin tether, Golgin45. We found that Golgin45 is a SUMOylated Golgin via SUMO1 under steady state condition. Upon heat shock stress, the Golgin enters the nucleus by interacting with Importin-ß2 and gets further modified by SUMO3. Importantly, SUMOylated Golgin45 appears to interact with PML and SUMO-deficient Golgin45 mutant functions as a dominant negative for PML-NB formation during heat shock stress, suppressing transcription of lipid metabolism genes. These results indicate that Golgin45 may play a role in heat stress response by transcriptional regulation of lipid metabolism genes in SUMOylation-dependent fashion.

GPS-SUMO 2.0: an updated online service for the prediction of SUMOylation sites and SUMO-interacting motifs.

Small ubiquitin-like modifiers (SUMOs) are tiny but important protein regulators involved in orchestrating a broad spectrum of biological processes, either by covalently modifying protein substrates or by noncovalently interacting with other proteins. Here, we report an updated server, GPS-SUMO 2.0, for the prediction of SUMOylation sites and SUMO-interacting motifs (SIMs). For predictor training, we adopted three machine learning algorithms, penalized logistic regression (PLR), a deep neural network (DNN), and a transformer, and used 52 404 nonredundant SUMOylation sites in 8262 proteins and 163 SIMs in 102 proteins. To further increase the accuracy of predicting SUMOylation sites, a pretraining model was first constructed using 145 545 protein lysine modification sites, followed by transfer learning to fine-tune the model. GPS-SUMO 2.0 exhibited greater accuracy in predicting SUMOylation sites than did other existing tools. For users, one or multiple protein sequences or identifiers can be input, and the prediction results are shown in a tabular list. In addition to the basic statistics, we integrated knowledge from 35 public resources to annotate SUMOylation sites or SIMs. The GPS-SUMO 2.0 server is freely available at https://sumo.biocuckoo.cn/. We believe that GPS-SUMO 2.0 can serve as a useful tool for further analysis of SUMOylation and SUMO interactions.

Unravelling the molecular interplay: SUMOylation, PML nuclear bodies and vascular cell activity in health and disease.

In the seemingly well-researched field of vascular research, there are still many underestimated factors and molecular mechanisms. In recent years, SUMOylation has become increasingly important. SUMOylation is a post-translational modification in which small ubiquitin-related modifiers (SUMO) are covalently attached to target proteins. Sites where these SUMO modification processes take place in the cell nucleus are PML nuclear bodies (PML-NBs) - multiprotein complexes with their essential main component and organizer, the PML protein. PML and SUMO, either alone or as partners, influence a variety of cellular processes, including regulation of transcription, senescence, DNA damage response and defence against microorganisms, and are involved in innate immunity and inflammatory responses. They also play an important role in maintaining homeostasis in the vascular system and in pathological processes leading to the development and progression of cardiovascular diseases. This review summarizes information about the function of SUMO(ylation) and PML(-NBs) in the human vasculature from angiogenesis to disease and highlights their clinical potential as drug targets.

Characterizing the Conformational Dynamics of Human SUMO2: Insights into its Interaction with Metal Ions and SIMs.

SUMO (Small Ubiquitin-like Modifiers) proteins are involved in a crucial post-translational modification commonly termed as SUMOylation. In this work, we have investigated the native-state conformational flexibility of human SUMO2 and its interaction with Cu2+ and Zn2+ ions using 15N-1H based 2D NMR spectroscopy. After SUMO1, SUMO2 is the most studied SUMO isoform in humans which shares 45 % and ~80 % similarity with SUMO1 in terms of sequence and structure, respectively. In this manuscript, we demonstrate that compared to SUMO1, several amino acids around the α1-helix region of SUMO2 access energetically similar near-native conformations. These conformations could play a crucial role in SUMO2's non-covalent interactions with SUMO interaction motifs (SIMs) on other proteins. The C-terminal of SUMO2 was found to bind strongly with Cu2+ ions resulting in a trimeric structure as observed by gel electrophoresis. This interaction seems to interfere in its non-covalent interaction with a V/I-x-V/I-V/I based SIM in Daxx protein.

Rhes, a striatal enriched protein, regulates post-translational small-ubiquitin-like-modifier (SUMO) modification of nuclear proteins and alters gene expression.

Rhes (Ras homolog enriched in the striatum), a multifunctional protein that regulates striatal functions associated with motor behaviors and neurological diseases, can shuttle from cell to cell via the formation of tunneling-like nanotubes (TNTs). However, the mechanisms by which Rhes mediates diverse functions remain unclear. Rhes is a small GTPase family member which contains a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes SUMO post-translational modification of proteins (SUMOylation) by promoting "cross-SUMOylation" of the SUMO enzyme SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). Nevertheless, the identity of the SUMO substrates of Rhes remains largely unknown. Here, by combining high throughput interactome and SUMO proteomics, we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene expression. Rhes increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, while decreasing SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 6 different lysine residues (K32, K110, K114, K120, K124, and K245). Furthermore, Rhes regulated the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide evidence for a previously undescribed role for Rhes in regulating the SUMOylation of nuclear targets and in orchestrating striatal gene expression via SUMOylation.

Small ubiquitin-like modifiers E3 ligases in plant stress.

Plants regularly encounter various environmental stresses such as salt, drought, cold, heat, heavy metals and pathogens, leading to changes in their proteome. Of these, a post-translational modification, SUMOylation is particularly significant for its extensive involvement in regulating various plant molecular processes to counteract these external stressors. Small ubiquitin-like modifiers (SUMO) protein modification significantly contributes to various plant functions, encompassing growth, development and response to environmental stresses. The SUMO system has a limited number of ligases even in fully sequenced plant genomes but SUMO E3 ligases are pivotal in recognising substrates during the process of SUMOylation. E3 ligases play pivotal roles in numerous biological and developmental processes in plants, including DNA repair, photomorphogenesis, phytohormone signalling and responses to abiotic and biotic stress. A considerable number of targets for E3 ligases are proteins implicated in reactions to abiotic and biotic stressors. This review sheds light on how plants respond to environmental stresses by focusing on recent findings on the role of SUMO E3 ligases, contributing to a better understanding of how plants react at a molecular level to such stressors.

Insights into the role of SUMO in regulating drought stress responses in pigeonpea (Cajanus cajan).

KEY MESSAGE: We have identified and analyzed 28 SUMO-pathway proteins from pigeonpea. Enhanced transcripts of pathway genes and increased SUMO conjugation under drought signifies the role of SUMO in regulating stress. Being a protein-rich and nutrient-dense legume crop, pigeonpea (Cajanus cajan) holds a vital position in a vegetarian meal. It is a resilient crop capable of striving in harsh climates and provides a means of subsistence to small-holding farmers. Nevertheless, extremes of water scarcity and drought conditions, especially during seedling and reproductive stages, remains a major issue severely impacting the growth and overall productivity of pigeonpea. Small ubiquitin-like modifier (SUMO), a post-translational modification system, plays a pivotal role in fortifying plants against stressful conditions by rapid reprogramming of molecular events. In this study, we have scanned the entire pigeonpea genome and identified 28 candidates corresponding to SUMO machinery components of pigeonpea. qRT-PCR analysis of different SUMO machinery genes validated their presence under natural conditions. The analysis of the promoters of identified SUMO machinery genes revealed the presence of abiotic stress-related cis-regulatory elements highlighting the potential involvement of the genes in abiotic stress responses. The transcript level analysis of selected SUMO machinery genes and global SUMO status of pigeonpea proteins in response to drought stress suggests an integral role of SUMO in regulating drought stress conditions in pigeonpea. Collectively, the work puts forward a detailed in silico analysis of pigeonpea SUMO machinery candidates and highlights the essential role of SUMOylation in drought stress responses. Being the first report on a pulse crop, the study will serve as a resource for devising strategies for counteracting drought stress in pigeonpea that can be further extended to other pulse crops.

SUMO and the DNA damage response.

The preservation of genome integrity requires specialised DNA damage repair (DDR) signalling pathways to respond to each type of DNA damage. A key feature of DDR is the integration of numerous post-translational modification signals with DNA repair factors. These modifications influence DDR factor recruitment to damaged DNA, activity, protein-protein interactions, and ultimately eviction to enable access for subsequent repair factors or termination of DDR signalling. SUMO1-3 (small ubiquitin-like modifier 1-3) conjugation has gained much recent attention. The SUMO-modified proteome is enriched with DNA repair factors. Here we provide a snapshot of our current understanding of how SUMO signalling impacts the major DNA repair pathways in mammalian cells. We highlight repeating themes of SUMO signalling used throughout DNA repair pathways including the assembly of protein complexes, competition with ubiquitin to promote DDR factor stability and ubiquitin-dependent degradation or extraction of SUMOylated DDR factors. As SUMO 'addiction' in cancer cells is protective to genomic integrity, targeting components of the SUMO machinery to potentiate DNA damaging therapy or exacerbate existing DNA repair defects is a promising area of study.

Depolymerization of SUMO chains induces slender to stumpy differentiation in T. brucei bloodstream parasites.

Trypanosoma brucei are protozoan parasites that cause sleeping sickness in humans and nagana in cattle. Inside the mammalian host, a quorum sensing-like mechanism coordinates its differentiation from a slender replicative form into a quiescent stumpy form, limiting growth and activating metabolic pathways that are beneficial to the parasite in the insect host. The post-translational modification of proteins with the Small Ubiquitin-like MOdifier (SUMO) enables dynamic regulation of cellular metabolism. SUMO can be conjugated to its targets as a monomer but can also form oligomeric chains. Here, we have investigated the role of SUMO chains in T. brucei by abolishing the ability of SUMO to polymerize. We have found that parasites able to conjugate only SUMO monomers are primed for differentiation. This was demonstrated for monomorphic lines that are normally unable to produce stumpy forms in response to quorum sensing signaling in mice, and also for pleomorphic cell lines in which stumpy cells were observed at unusually low parasitemia levels. SUMO chain mutants showed a stumpy compatible transcriptional profile and better competence to differentiate into procyclics. Our study indicates that SUMO depolymerization may represent a coordinated signal triggered during stumpy activation program.

Abnormal protein SUMOylation in liver disease: novel target for therapy.

SUMOylation is an important protein post-translational modification (PTM) process, in which the small ubiquitin-like modifier (SUMO) protein covalently binds to the target protein and regulates stability, subcellular localization, and protein-protein interaction of the target protein. Protein SUMOylation exerts crucial regulatory function in the liver, and its abnormalities are associated with various liver-related disease processes. This review focuses on the biological functions of protein SUMOylation in liver-related diseases in recent years, summarizes the molecular mechanisms of SUMOylation in the replication of hepatitis viruses and the occurrence of hepatocellular carcinoma, and discusses the significance of SUMOylation in liver-related disorders, which is essential for understanding liver biological processes and formulating therapeutic strategies.

E3 SUMO ligase SIZ1 splicing variants localize and function according to external conditions.

SIZ1 (SAP and MIZ1) is a member of the Siz/PIAS-type RING family of E3 SUMO (small ubiquitin-related modifier) ligases that play key roles in growth, development, and stress responses in plant and animal systems. Nevertheless, splicing variants of SIZ1 have not yet been characterized. Here, we identified four splicing variants of Arabidopsis (Arabidopsis thaliana) SIZ1, which encode three different protein isoforms. The SIZ1 gene encodes an 873-amino acid (aa) protein. Among the four SIZ1 splicing variants (SSVs), SSV1 and SSV4 encode identical 885 aa proteins; SSV2 encodes an 832 aa protein; and SSV3 encodes an 884 aa protein. SSV2 mainly localized to the plasma membrane, whereas SIZ1, SSV1/SSV4, and SSV3 localized to the nucleus. Interestingly, SIZ1 and all SSVs exhibited similar E3 SUMO ligase activities and preferred SUMO1 and SUMO2 for their E3 ligase activity. Transcript levels of SSV2 were substantially increased by heat treatment, while those of SSV1, SSV3, and SSV4 transcripts were unaffected by various abiotic stresses. SSV2 directly interacted with and sumoylated cyclic nucleotide-gated ion channel 6 (CNGC6), a positive thermotolerance regulator, enhancing the stability of CNGC6. Notably, transgenic siz1-2 mutants expressing SSV2 exhibited greater heat stress tolerance than wild-type plants, whereas those expressing SIZ1 were sensitive to heat stress. Furthermore, transgenic cngc6 plants overaccumulating a mutated mCNGC6 protein (K347R, a mutation at the sumoylation site) were sensitive to heat stress, similar to the cngc6 mutants, while transgenic cngc6 plants overaccumulating CNGC6 exhibited restored heat tolerance. Together, we propose that alternative splicing is an important mechanism that regulates the function of SSVs during development or under adverse conditions, including heat stress.

Molecular Organization and Regulation of the Mammalian Synapse by the Post-Translational Modification SUMOylation.

Neurotransmission occurs within highly specialized compartments forming the active synapse where the complex organization and dynamics of the interactions are tightly orchestrated both in time and space. Post-translational modifications (PTMs) are central to these spatiotemporal regulations to ensure an efficient synaptic transmission. SUMOylation is a dynamic PTM that modulates the interactions between proteins and consequently regulates the conformation, the distribution and the trafficking of the SUMO-target proteins. SUMOylation plays a crucial role in synapse formation and stabilization, as well as in the regulation of synaptic transmission and plasticity. In this review, we summarize the molecular consequences of this protein modification in the structural organization and function of the mammalian synapse. We also outline novel activity-dependent regulation and consequences of the SUMO process and explore how this protein modification can functionally participate in the compartmentalization of both pre- and post-synaptic sites.

SUMO modifies GßL and mediates mTOR signaling.

The mechanistic target of rapamycin (mTOR) signaling is influenced by multiple regulatory proteins and post-translational modifications; however, underlying mechanisms remain unclear. Here, we report a novel role of small ubiquitin-like modifier (SUMO) in mTOR complex assembly and activity. By investigating the SUMOylation status of core mTOR components, we observed that the regulatory subunit, GßL (G protein ß-subunit-like protein, also known as mLST8), is modified by SUMO1, 2, and 3 isoforms. Using mutagenesis and mass spectrometry, we identified that GßL is SUMOylated at lysine sites K86, K215, K245, K261, and K305. We found that SUMO depletion reduces mTOR-Raptor (regulatory protein associated with mTOR) and mTOR-Rictor (rapamycin-insensitive companion of mTOR) complex formation and diminishes nutrient-induced mTOR signaling. Reconstitution with WT GßL but not SUMOylation-defective KR mutant GßL promotes mTOR signaling in GßL-depleted cells. Taken together, we report for the very first time that SUMO modifies GßL, influences the assembly of mTOR protein complexes, and regulates mTOR activity.

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.

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

BACKGROUND: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.