Sumoylation in Development and Differentiation.

Tissue morphogenesis is a fascinating aspect of both developmental biology and regeneration of certain adult organs, and timely control of cellular differentiation is a key to these processes. During development, events interrupting cellular differentiation and leading to organ failure are embryonic lethal; likewise, perturbation of differentiation in regenerating tissues leads to dysfunction and disease. At the molecular level, cellular differentiation is orchestrated by a well-coordinated cascade of transcription factors (TFs) and chromatin remodeling complexes that drive gene expression. Altering the localization, stability, or activity of these regulatory elements can affect the sequential organization of the gene expression program and result in failed or abnormal tissue development. An accumulating body of evidence shows that the sumoylation system is a critical modulator of these regulatory cascades. For example, inhibition of the sumoylation system during embryogenesis causes lethality and/or severe abnormalities from invertebrates to mammals. Mechanistically, it is now known that many of the TFs and components of chromatin remodeling complexes that are critical for development and differentiation are targets for SUMO modification, though the specific functional consequences of the modifications remain uncharacterized in many cases. This chapter will address several of the models systems that have been examined for the role of sumoylation in differentiation and development. Understanding the profound regulatory role of SUMO in different tissues should lead not only to a better understanding of developmental biology, stem cell linage control, and the mechanisms of cellular differentiation, but may also lead to the identification of new targets for drug therapy and/or therapeutic manipulation of damaged organs and tissues.

Viral Interplay with the Host Sumoylation System.

Viruses have evolved elaborate means to regulate diverse cellular pathways in order to create a cellular environment that facilitates viral survival and reproduction. This includes enhancing viral macromolecular synthesis and assembly, as well as preventing antiviral responses, including intrinsic, innate, and adaptive immunity. There are numerous mechanisms by which viruses mediate their effects on the host cell, and this includes targeting various cellular post-translational modification systems, including sumoylation. The wide-ranging impact of sumoylation on cellular processes such as transcriptional regulation, apoptosis, stress response, and cell cycle control makes it an attractive target for viral dysregulation. To date, proteins from both RNA and DNA virus families have been shown to be modified by SUMO conjugation, and this modification appears critical for viral protein function. More interestingly, members of the several viral families have been shown to modulate sumoylation, including papillomaviruses, adenoviruses , herpesviruses, orthomyxoviruses, filoviruses , and picornaviruses . This chapter will focus on mechanisms by which sumoylation both impacts human viruses and is used by viruses to promote viral infection and disease.

The role of ubiquitin and ubiquitin-like modification systems in papillomavirus biology.

Human papillomaviruses (HPVs) are small DNA viruses that are important etiological agents of a spectrum of human skin lesions from benign to malignant. Because of their limited genome coding capacity they express only a small number of proteins, only one of which has enzymatic activity. Additionally, the HPV productive life cycle is intimately tied to the epithelial differentiation program and they must replicate in what are normally non-replicative cells, thus, these viruses must reprogram the cellular environment to achieve viral reproduction. Because of these limitations and needs, the viral proteins have evolved to co-opt cellular processes primarily through protein-protein interactions with critical host proteins. The ubiquitin post-translational modification system and the related ubiquitin-like modifiers constitute a widespread cellular regulatory network that controls the levels and functions of thousands of proteins, making these systems an attractive target for viral manipulation. This review describes the interactions between HPVs and the ubiquitin family of modifiers, both to regulate the viral proteins themselves and to remodel the host cell to facilitate viral survival and reproduction.

Cell culture assay for transient replication of human and animal papillomaviruses.

This unit contains protocols for evaluation of replication functionality of papillomavirus genomes or subgenomic fragments. Replication is measured after transient cotransfection of the genome (or subgenomic fragment) with expression vectors encoding the viral E1 and E2 proteins. Input DNA is methylated at the adenine of GATC sequences by propagation in E. coli. DNA that replicates in mammalian cells will lose the adenine methylation and become DpnI-resistant, while residual methylated input DNA will remain DpnI-sensitive. After transfection, DNA extraction, and DpnI digestion, replicated DNA can be detected by Southern blotting as a full-length plasmid, since it is resistant to digestion. This assay can be used to map the genomic location of a functional origin or to evaluate replication activity of mutations in either the origin DNA sequences or the E1 or E2 proteins.

Analysis of global sumoylation changes occurring during keratinocyte differentiation.

Sumoylation is a highly dynamic process that plays a role in a multitude of processes ranging from cell cycle progression to mRNA processing and cancer. A previous study from our lab demonstrated that SUMO plays an important role in keratinocyte differentiation. Here we present a new method of tracking the sumoylation state of proteins by creating a stably transfected HaCaT keratinocyte cell line expressing an inducible SNAP-SUMO3 protein. The SNAP-tag allows covalent fluorescent labeling that is denaturation resistant. When combined with two-dimensional gel electrophoresis, the SNAP-tag technology provides direct visualization of sumoylated targets and can be used to follow temporal changes in the global cohort of sumoylated proteins during dynamic processes such as differentiation. HaCaT keratinocyte cells expressing SNAP-SUMO3 displayed normal morphological and biochemical features that are consistent with typical keratinocyte differentiation. SNAP-SUMO3 also localized normally in these cells with a predominantly nuclear signal and some minor cytoplasmic staining, consistent with previous reports for untagged SUMO2/3. During keratinocyte differentiation the total number of proteins modified by SNAP-SUMO3 was highest in basal cells, decreased abruptly after induction of differentiation, and slowly rebounded beginning between 48 and 72 hours as differentiation progressed. However, within this overall trend the pattern of change for individual sumoylated proteins was highly variable with both increases and decreases in amount over time. From these results we conclude that sumoylation of proteins during keratinocyte differentiation is a complex process which likely reflects and contributes to the biochemical changes that drive differentiation.

HPV E6 proteins target Ubc9, the SUMO conjugating enzyme.

The human papillomavirus oncogenic protein, E6, interacts with a number of cellular proteins, and for some targets, E6 directs their degradation through the ubiquitin-proteasome pathway. Post-translational modification with ubiquitin-like modifiers, such as SUMO, also influences protein activities, protein-protein interactions, and protein stability. We report that the high risk HPVE6 proteins reduce the intracellular quantity of the sole SUMO conjugation enzyme, Ubc9, concomitant with decreased host sumoylation. E6 did not significantly influence transcription of Ubc9, indicating that the effects were likely at the protein level. Consistent with typical E6-mediated proteasomal degradation, E6 bound to Ubc9 in vitro, and required E6AP for reduction of Ubc9 levels. Under stable E6 expression conditions in differentiating keratinocytes there was a decrease in Ubc9 and a loss of numerous sumoylated targets indicating a significant perturbation of the normal sumoylation profile. While E6 is known to inhibit PIASy, a SUMO ligase, our results suggest that HPV E6 also targets the Ubc9 protein to modulate host cell sumoylation, suggesting that the sumoylation system may be an important target during viral reproduction and possibly the subsequent development of cervical cancer.

Common importin alpha specificity for papillomavirus E2 proteins.

Papillomaviruses infect keratinocytes and their reproduction is tied to differentiation of the skin. The E2 protein of papillomaviruses is a multifunctional early protein that binds specifically to the viral DNA to regulate genome transcription, replication, and segregation. All of these are nuclear events that require specific transport of E2 into the host nucleus. Nuclear localization signal (NLS) sequences have been mapped for several E2 proteins, and these sequences resemble motifs that interact with cellular transport adaptor molecules termed alpha importins. To determine which importins could carry E2 proteins, in vitro binding studies were performed with three different E2 proteins and the five ubiquitous alpha importins. The E2 proteins preferentially interacted with alpha importins 3 and 5, and showed very weak or no interaction with the other three widely expressed alpha importins (alpha1, alpha 4, and alpha 7). While all five alpha importins appear to be constitutively expressed in keratinocytes, during differentiation of a human keratinocyte line (HaCaT) we observed a specific increase in expression of alphas 3 and 5. This differentiation-specific increase in alpha 3 and alpha 5 expression suggests that preferential usage of these two importins by E2 may facilitate E2 nuclear uptake during terminal differentiation.

Host cell sumoylation level influences papillomavirus E2 protein stability.

The stability of papillomavirus E2 proteins is regulated by proteasomal degradation, and regulation of degradation could contribute to the higher expression levels of E2 proteins observed in suprabasal layers of differentiated skin. We have recently shown that the E2 proteins are modified by sumoylation [Wu Y-C, Roark AA, Bian X-L, Wilson, VG (2008) Virol 378:329-338], and that sumoylation levels are up-regulated during keratinocyte differentiation [Deyrieux AF, Rosas-Acosta G, Ozbun MA, Wilson VG (2007) J Cell Sci 120:125-136]. These observations, coupled with the known ability of sumoylation to prevent proteasomal degradation of certain proteins, suggested that this modification might contribute to stabilizing E2 proteins in suprabasal keratinocytes. Conditions that increased overall sumoylation were found to increase the intracellular amounts of the HPV11, 16, and 18 E2 proteins. No effect of sumoylation was seen on E2 transcripts, and the increased levels of E2 proteins resulted from a greatly increased half-life for the E2 proteins. In vitro studies confirmed that sumoylation could block the proteasomal degradation of the 16E2 protein. Interestingly, this stabilization effect was indirect as it did not require sumoylation of 16E2 itself and must be acting through sumoylation of a cellular target(s). This sumoylation-dependent, indirect stabilization of E2 proteins is a novel process that may couple E2 levels to changes in the cellular environment. Specifically, our results suggest that the levels of papillomavirus E2 protein could be up-regulated in differentiating keratinocytes in response to the increased overall sumoylation that accompanies differentiation.

Signalling of the BCR is regulated by a lipid rafts-localised transcription factor, Bright.

Regulation of BCR signalling strength is crucial for B-cell development and function. Bright is a B-cell-restricted factor that complexes with Bruton's tyrosine kinase (Btk) and its substrate, transcription initiation factor-I (TFII-I), to activate immunoglobulin heavy chain gene transcription in the nucleus. Here we show that a palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. After BCR ligation, Bright transiently interacts with sumoylation enzymes, blocks calcium flux and phosphorylation of Btk and TFII-I and is then discharged from lipid rafts as a Sumo-I-modified form. The resulting lipid raft concentration of Bright contributes to the signalling threshold of B cells, as their sensitivity to BCR stimulation decreases as the levels of Bright increase. Bright regulates signalling independent of its role in IgH transcription, as shown by specific dominant-negative titration of rafts-specific forms. This study identifies a BCR tuning mechanism in lipid rafts that is regulated by differential post-translational modification of a transcription factor with implications for B-cell tolerance and autoimmunity.

Mice overexpressing murine oncostatin M (OSM) exhibit changes in hematopoietic and other organs that are distinct from those of mice overexpressing human OSM or bovine OSM.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) belong to the interleukin-6 family of cytokines. The authors' previous in vitro work demonstrated that in mouse cells mouse OSM (mOSM) signals through a heterodimeric receptor complex incorporating the mOSM-specific receptor mOSMRbeta while human OSM (hOSM) and bovine OSM (bOSM) use the mouse LIF receptor mLIFRbeta rather than mOSMRbeta. These in vitro data suggest that prior studies in mouse systems with hOSM or bOSM (the usual molecules used in early studies) reflect LIF rather than OSM biology. The current work assessed whether or not this divergence in actions among these three OSMs also occurs in vivo in mouse models. Adult female (C57BL/6J x DBA/2J) F(1) mice were engineered to stably overexpress mOSM, hOSM, or bOSM by retrovirus-mediated gene transfer (n = 10 or more per group). After 4 weeks, molecular and hematologic profiles and anatomic phenotypes in multiple organs were assessed by standard techniques. Animals overexpressing either hOSM or bOSM had an identical phenotype resembling that associated with LIF activation, including significant hematologic abnormalities (anemia, neutrophilia, lymphopenia, eosinopenia, and thrombocytosis); weight loss; profound enlargement (lymph node, spleen) and/or structural reorganization (lymph node, spleen, thymus) of lymphoid organs; and severe osteosclerosis. In contrast, mice overexpressing mOSM did not develop hematologic changes, weight loss, or osteosclerosis and exhibited more modest and anatomically distinct restructuring of lymphoid organs. These data indicate that activities imputed to OSM and the mOSMRbeta signaling pathway using in vitro and in vivo mouse experimental systems are unique to mOSM.

[Management of a diabetic foot ulcer]. / Conduite à tenir face à une plaie du pied chez un diabétique.

A chronic diabetic foot ulcer requires a search for the etiology. The three main causes to search for are poor off-loading compliance, osteomyelitis, and peripheral vascular disease. The level of severity is measured with the U.T. classification and the level of infection with the classification of the International Consensus on the Diabetic Foot. Peripheral vascular disease must be precisely evaluated by Doppler ultrasound, which describes all the arteries of the lower limb. Angiography is required only in case of revascularization. Treatment of the ulcer includes strict off-loading, topical treatment, optimal treatment of hyperglycemia, and antibiotic therapy on a case-by-case basis for osteomyelitis and/or, angioplasty or by-pass procedures. Osteomyelitis can be treated by associating conservative surgery, antibiotic therapy, and off-loading. No amputation, even of one toe, must be done without a previous vascular check-up. Off-loading of the ulcer must be regularly checked. Poor off-loading compliance must be systematically investigated if the ulcer worsens or healing is delayed.

Modification of papillomavirus E2 proteins by the small ubiquitin-like modifier family members (SUMOs).

Papillomavirus E2 proteins are critical regulatory proteins that function in replication, genome segregation, and viral transcription, including control of expression of the viral oncogenes, E6 and E7. Sumoylation is a post-translational modification that has been shown to target and modulate the function of many transcription factors, and we now demonstrate that E2 proteins are sumoylated. Both bovine and human papillomavirus E2 proteins bind to the SUMO conjugation enzyme, Ubc9, and using in vitro and E. coli sumoylation systems, these E2 proteins were readily modified by SUMO proteins. In vivo experiments further confirmed that E2 can be sumoylated by SUMO1, SUMO2, or SUMO3. Mapping studies identified lysine 292 as the principal residue for covalent conjugation of SUMO to HPV16 E2, and a lysine 292 to arginine mutant showed defects for both transcriptional activation and repression. The expression levels, intracellular localization, and the DNA-binding activity of HPV16 E2 were unchanged by this K292R mutation, suggesting that the transcriptional defect reflects a functional contribution by sumoylation at this residue. This study provides evidence that sumoylation has a role in the regulation of papillomavirus E2, and identifies a new mechanism for the modulation of E2 function at the post-translational level.

Ubiquitin proteolytic system: focus on SUMO.

The small ubiquitin-like modifier proteins (Smt3 in yeast and SUMOs 1-4 in vertebrates) are members of the ubiquitin super family. Like ubiquitin, the SUMOs are protein modifiers that are covalently attached to the epsilon-amino group of lysine residues in the substrates. The application of proteomics to the SUMO field has greatly expanded both the number of known targets and the number of identified target lysines. As new refinements of proteomic techniques are developed and applied to sumoylation, an explosion of novel data is likely in the next 5 years. This ability to examine sumoylated proteins globally, rather than individually, will lead to new insights into both the functions of the individual SUMO types, and how dynamic changes in overall sumoylation occur in response to alterations in cellular environment. In addition, there is a growing appreciation for the existence of cross-talk mechanisms between the sumoylation and ubiquitinylation processes. Rather than being strictly parallel, these two systems have many points of intersection, and it is likely that the coordination of these two systems is a critical contributor to the regulation of many fundamental cellular events.

Identification of a nuclear export signal sequence for bovine papillomavirus E1 protein.

Recent studies have demonstrated nuclear export by papillomavirus E1 proteins, but the requisite export sequence(s) for bovine papillomavirus (BPV) E1 were not defined. In this report we identify three functional nuclear export sequences (NES) present in BPV E1, with NES2 being the strongest in reporter assays. Nuclear localization of BPV1 E1 was modulated by over- or under-expression of CRM1, the major cellular exportin, and export was strongly reduced by the CRM1 inhibitor, Leptomycin B, indicating that E1 export occurs primarily through a CRM1-dependent process. Consistent with the in vivo functional results, E1 bound CRM1 in an in vitro pull-down assay. In addition, sumoylated E1 bound CRM1 more effectively than unmodified E1, suggesting that E1 export may be regulated by SUMO modification. Lastly, an E1 NES2 mutant accumulated in the nucleus to a greater extent than wild-type E1, yet was defective for viral origin replication in vivo. However, NES2 exhibited no intrinsic replication defect in an in vitro replication assay, implying that nucleocytoplasmic shuttling may be required to maintain E1 in a replication competent state.

Nuclear import of bovine papillomavirus type 1 E1 protein is mediated by multiple alpha importins and is negatively regulated by phosphorylation near a nuclear localization signal.

Papillomavirus DNA replication occurs in the nucleus of infected cells and requires the viral E1 protein, which enters the nuclei of host epithelial cells and carries out enzymatic functions required for the initiation of viral DNA replication. In this study, we investigated the pathway and regulation of the nuclear import of the E1 protein from bovine papillomavirus type 1 (BPV1). Using an in vitro binding assay, we determined that the E1 protein interacted with importins alpha3, alpha4, and alpha5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified E1 protein was effectively imported into the nucleus of digitonin-permeabilized HeLa cells after incubation with importin alpha3, alpha4, or alpha5 and other necessary import factors. We also observed that in vitro binding of E1 protein to all three alpha importins was significantly decreased by the introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated E1 protein failed to enter the nucleus of digitonin-permeabilized HeLa cells in vitro. Likewise, the pseudophosphorylation mutant showed aberrant intracellular localization in vivo and accumulated primarily on the nuclear envelope in transfected HeLa cells, while the corresponding alanine replacement mutant displayed the same cellular location pattern as wild-type E1 protein. Collectively, our data demonstrate that BPV1 E1 protein can be transported into the nucleus by more than one importin alpha and suggest that E1 phosphorylation by host cell kinases plays a regulatory role in modulating E1 nucleocytoplasmic localization. This phosphoregulation of nuclear E1 protein uptake may contribute to the coordination of viral replication with keratinocyte proliferation and differentiation.

Percutaneous transluminal angioplasty in severe diabetic foot ischemia: outcomes and prognostic factors.

OBJECTIVES: To evaluate the outcomes of severe ischemic diabetic foot ulcers for which percutaneous transluminal angioplasty (PTA) was considered as the first-line vascular procedure. Factors associated with successful PTA were sought. RESEARCH DESIGN AND METHODS: In 32 consecutive diabetic patients with foot ulcers and severe limb ischemia, PTA was performed if feasible; if not, primary bypass grafting was done when feasible. All patients were followed until healing or for at least one year. Patients with worsening ulcers after PTA underwent bypass grafting. Clinical and angiographic factors influencing outcomes after PTA were sought by univariate and multivariate analysis. RESULTS: PTA was done in 25 of the 32 (78%) patients, and considered clinically successful in 13 (52%). After 1 year, the healing rate was 70% and the limb salvage rate 90%. Successful PTA was significantly associated with a higher post-PTA transcutaneous oxygen pressure (P = 0.03) and presence of at least one patent pedal vessel (P = 0.03) in the univariate analysis; only a patent pedal vessel was significant in the multivariate analysis. CONCLUSION: Primary PTA in diabetic patients with severe ischemic foot ulcers provides similar outcomes to usual results obtained in severe ischemia in absence of diabetes. The presence of one patent pedal vessel on arteriography before PTA is the best prognostic factor.

Wrestling with SUMO in a new arena.

Sumoylation is a widespread posttranslational modification thought to affect primarily nuclear proteins, especially transcription factors for which sumoylation usually results in repression of their transactivational function. Recent proteomics studies have greatly expanded the cadre of known SUMO substrates, and an increasing number of cytoplasmic proteins have been identified as SUMO targets. However, very few of these cytosolic proteins have been evaluated for the functional consequences of sumoylation. Rajan et al. now demonstrate that the activity of an integral cytoplasmic membrane channel-forming protein, K2P1, is completely abrogated by sumoylation at a single lysine residue on the cytoplasmic tail. This is the first report of a plasma membrane protein as a SUMO substrate and explains the long-standing inability to demonstrate functionality of K2P1. Apparently, K2P1 is stoichiometrically sumoylated under most cellular conditions, so it is constitutively inactive until desumoylated. These observations raise several intriguing questions, including: How and where does K2P1 become sumoylated? Why, unlike most known substrates, is K2P1 so efficiently sumoylated? and, What are the signals and SUMO proteases that trigger K2P1 desumoylation? But most importantly, the report by Rajan et al. expands the functional roles attributed to sumoylation into the new arena of membrane protein functional regulation and suggests that similar mechanisms may regulate the function of other pore proteins.

Cell culture assay for transient replication of human and animal papillomaviruses.

This unit contains protocols for evaluation of replication functionality of papillomavirus genomes or subgenomic fragments. Replication is measured after transient cotransfection of the genome (or subgenomic fragment) with expression vectors encoding the viral E1 and E2 proteins. Input DNA is methylated at the adenine of GATC sequences by propagation in E. coli. DNA that replicates in mammalian cells will lose the adenine methylation and become DpnI-resistant, while residual methylated input DNA will remain DpnI-sensitive. After transfection, DNA extraction, and DpnI digestion, replicated DNA can be detected by Southern blotting as a full-length plasmid, since it is resistant to digestion. This assay can be used to map the genomic location of a functional origin or to evaluate replication activity of mutations in either the origin DNA sequences or the E1 or E2 proteins.

A universal strategy for proteomic studies of SUMO and other ubiquitin-like modifiers.

Post-translational modification by the conjugation of small ubiquitin-like modifiers is an essential mechanism to affect protein function. Currently, only a limited number of substrates are known for most of these modifiers, thus limiting our knowledge of their role and relevance for cellular physiology. Here, we report the development of a universal strategy for proteomic studies of ubiquitin-like modifiers. This strategy involves the development of stable transfected cell lines expressing a double-tagged modifier under the control of a tightly negatively regulated promoter, the induction of the expression and conjugation of the tagged modifier to cellular proteins, the tandem affinity purification of the pool of proteins covalently modified by the tagged modifier, and the identification of the modified proteins by LC and MS. By applying this methodology to the proteomic analysis of SUMO-1 and SUMO-3, we determined that SUMO-1 and SUMO-3 are stable proteins exhibiting half-lives of over 20 h, demonstrated that sumoylation with both SUMO-1 and SUMO-3 is greatly stimulated by MG-132 and heat shock treatment, demonstrated the preferential usage of either SUMO-1 or SUMO-3 for some known SUMO substrates, and identified 122 putative SUMO substrates of which only 27 appeared to be modified by both SUMO-1 and SUMO-3. This limited overlapping in the subset of proteins modified by SUMO-1 and SUMO-3 supports that the SUMO paralogues are likely to be functionally distinct. Three of the novel putative SUMO substrates identified, namely the polypyrimidine tract-binding protein-associated splicing factor PSF, the structural microtubular component alpha-tubulin, and the GTP-binding nuclear protein Ran, were confirmed as authentic SUMO substrates. The application of this universal strategy to the identification of the pool of cellular substrates modified by other ubiquitin-like modifiers will dramatically increase our knowledge of the biological role of the different ubiquitin-like conjugations systems in the cell.

Proteins of the PIAS family enhance the sumoylation of the papillomavirus E1 protein.

Sumoylation of the papillomavirus (PV) origin binding helicase E1 protein is critical for its function. Consequently, factors modulating the sumoylation of E1 could ultimately alter the outcome of a papillomavirus infection. We investigated the role played by phosphorylation and two known SUMO E3 ligases, RanBP2 and PIAS proteins, on the sumoylation of E1. E1 sumoylation was unaffected by phosphorylation as both wild-type and pseudo-phosphorylation mutants of BPV E1 exhibited similar sumoylation profiles. RanBP2 bound to BPV E1, but not to HPV11 E1, and lacked sumoylation enhancing activity for either E1. In contrast, proteins of the PIAS family (except PIASy) bound to both BPV and HPV11 E1 and stimulated their sumoylation. The structural integrity of the RING finger domain of the PIAS proteins was required for their E3 SUMO ligase activity on PV E1 sumoylation but was dispensable for their PV E1 binding activity. Miz1, the PIAS protein exerting the strongest E1 sumoylation enhancing activity, favored SUMO1 versus SUMO2 as the modifier and was shown to be transcribed in a keratinocyte cell line. This study indicates PIAS proteins as possible modulators of PV E1 sumoylation during papillomavirus infections.