SUMO proteins are ubiquitin-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair, and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. Here we report isolation and characterization of SUMO1-and SUMO2-binding motifs. Using yeast two-hybrid system, bioinformatics, and NMR spectroscopy we define a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a ␤-strand that could bind in parallel or antiparallel orientation to the ␤ 2 -strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions. SUMO proteins are small ubiquitin (Ub)5 -related modifiers that become conjugated to cellular substrates and regulate diverse cellular processes including cell cycle progression, intracellular trafficking, transcription, and DNA repair (1-3). Like Ub, a SUMO protein is covalently attached to target proteins through an isopeptide bond by a mechanism similar to that of ubiquitination, which involves E1, E2, and E3 enzymes (4). In mammals, three SUMO paralogues are commonly expressed: SUMO1 shares about 45% identity to SUMO2 and SUMO3, while SUMO2 and SUMO3 are 96% identical to each other (2, 5).The structures of all three SUMO paralogues resemble the globular and compact Ub-like fold (6, 7). The differences of SUMO1 and SUMO2 are mostly found in the second ␤-strand and the ␣-helix of both proteins (7). In cells, different SUMO paralogues appear to share common properties but also have some distinct functions. For example, the promyelocytic leukemia protein is conjugated to all three SUMO paralogs (8, 9), whereas RanGAP1 is preferentially modified with SUMO1 (10) and topoisomerase II with SUMO2/3 during mitosis (11). Furthermore, the distribution of the SUMO paralogues within cells seems to be different. SUMO1 is uniquely found within the nucleoli, the nuclear envelope, and cytoplasmic foci, whereas SUMO2/3 are accrued on chromosomes at an earlier point in the nuclear reformation process (12). Interestingly, there is a larger pool of free, non-conjugated SUMO2/3 than of SUMO1 (10).In addition to targeting different substrate proteins, the functional properties of SUMO isoforms in vivo might also reflect their ability to mediate distinct protein-protein interactions. Indeed, recent studies have shown that SUMO paralogues can promote non-covalent binding to other proteins containing specific motifs that recognize SUMO paralogues. Minty and coworkers defined a Ser-Xaa-Ser motif surrounded by hydrophobic and acidic amino acids as a SUMO-interacting motif (SIM) (13). Biophysical studies of the SIM in PIAS revealed that the small hydrophobic region is an essential determinant of SUMO recognition (14). Moreover...

Ubiquitin and ubiquitin-like proteins (Ubls) are signalling messengers that control many cellular functions, such as cell proliferation, apoptosis, the cell cycle and DNA repair. It is becoming apparent that the deregulation of ubiquitin pathways results in the development of human diseases, including many types of tumours. Here we summarize the common principles and specific features of ubiquitin and Ubls in the regulation of cancer-relevant pathways, and discuss new strategies to target ubiquitin signalling in drug discovery.

Ubiquitin (Ub)-binding domains (UBDs) are key elements in conveying Ub-based cellular signals. UBD-containing proteins interact with ubiquitinated targets and control numerous biological processes. They themselves undergo UBD-dependent monoubiquitination, which promotes intramolecular binding of the UBD to the attached Ub and leads to their inactivation. Here, we report that, in contrast to the established ubiquitination pathway, the presence of UBDs allows the ubiquitination of host proteins independently of E3 ligases. UBDs of different types, including UBA, UIM, UBM, NFZ, and UBZ, can directly cooperate with Ub-charged E2 enzymes to promote monoubiquitination. Using FRET and siRNA technologies, we verify that Ub-loaded E2 and substrates interact in cells and that E2 enzymes are essential for their monoubiquitination in vivo. This modification is mechanistically and functionally distinct from E3-mediated and growth factor-dependent monoubiquitination.

TANK-binding kinase 1 (TBK1/NAK/T2K) and I-jB Kinase (IKK-i/IKK-e) play important roles in the regulation of interferon (IFN)-inducible genes during the immune response to bacterial and viral infections. Cell stimulation with ssRNA virus, dsDNA virus or gram-negative bacteria leads to activation of TBK1 or IKK-i, which in turn phosphorylates the transcription factors, IFN-regulatory factor (IRF) 3 and IRF7, promoting their translocation in the nucleus. To understand the molecular basis of activation of TBK1, we analyzed the sequence of TBK1 and IKK-i and identified a ubiquitin-like domain (ULD) adjacent to their kinase domains. Deletion or mutations of the ULD in TBK1 or IKK-i impaired activation of respective kinases, failed to induce IRF3 phosphorylation and nuclear localization and to activate IFN-b or RANTES promoters. The importance of the ULD of TBK1 in LPS-or poly(I:C)-stimulated IFN-b production was demonstrated by reconstitution experiments in TBK1-IKK-i-deficient cells. We propose that the ULD is a regulatory component of the TBK1/IKK-i kinases involved in the control of the kinase activation, substrate presentation and downstream signaling pathways.

Neuroinflammation is a pathophysiological hallmark of multiple sclerosis and has a close mechanistic link to neurodegeneration. Although this link is potentially targetable, robust translatable models to reliably quantify and track neuroinflammation in both mice and humans are lacking. The choroid plexus (ChP) plays a pivotal role in regulating the trafficking of immune cells from the brain parenchyma into the cerebrospinal fluid (CSF) and has recently attracted attention as a key structure in the initiation of inflammatory brain responses. In a translational framework, we here address the integrity and multidimensional characteristics of the ChP under inflammatory conditions and question whether ChP volumes could act as an interspecies marker of neuroinflammation that closely interrelates with functional impairment. Therefore, we explore ChP characteristics in neuroinflammation in patients with multiple sclerosis and in two experimental mouse models, cuprizone diet-related demyelination and experimental autoimmune encephalomyelitis. We demonstrate that ChP enlargement—reconstructed from MRI—is highly associated with acute disease activity, both in the studied mouse models and in humans. A close dependency of ChP integrity and molecular signatures of neuroinflammation is shown in the performed transcriptomic analyses. Moreover, pharmacological modulation of the blood–CSF barrier with natalizumab prevents an increase of the ChP volume. ChP enlargement is strongly linked to emerging functional impairment as depicted in the mouse models and in multiple sclerosis patients. Our findings identify ChP characteristics as robust and translatable hallmarks of acute and ongoing neuroinflammatory activity in mice and humans that could serve as a promising interspecies marker for translational and reverse-translational approaches.

Chronic disability in multiple sclerosis is linked to neuroaxonal degeneration. 4-aminopyridine (4-AP) is used and licensed as a symptomatic treatment to ameliorate ambulatory disability in multiple sclerosis. The presumed mode of action is via blockade of axonal voltage gated potassium channels, thereby enhancing conduction in demyelinated axons. In this study, we provide evidence that in addition to those symptomatic effects, 4-AP can prevent neuroaxonal loss in the CNS. Using in vivo optical coherence tomography imaging, visual function testing and histologic assessment, we observed a reduction in retinal neurodegeneration with 4-AP in models of experimental optic neuritis and optic nerve crush. These effects were not related to an anti-inflammatory mode of action or a direct impact on retinal ganglion cells. Rather, histology and in vitro experiments indicated 4-AP stabilization of myelin and oligodendrocyte precursor cells associated with increased nuclear translocation of the nuclear factor of activated T cells. In experimental optic neuritis, 4-AP potentiated the effects of immunomodulatory treatment with fingolimod. As extended release 4-AP is already licensed for symptomatic multiple sclerosis treatment, we performed a retrospective, multicentre optical coherence tomography study to longitudinally compare retinal neurodegeneration between 52 patients on continuous 4-AP therapy and 51 matched controls. In line with the experimental data, during concurrent 4-AP therapy, degeneration of the macular retinal nerve fibre layer was reduced over 2 years. These results indicate disease-modifying effects of 4-AP beyond symptomatic therapy and provide support for the design of a prospective clinical study using visual function and retinal structure as outcome parameters.

Background: Retinal optical coherence tomography (OCT) is a clinical and research tool in multiple sclerosis, where it has shown significant retinal nerve fiber (RNFL) and ganglion cell (RGC) layer thinning, while postmortem studies have reported RGC loss. Although retinal pathology in experimental autoimmune encephalomyelitis (EAE) has been described, comparative OCT studies among EAE models are scarce. Furthermore, the best practices for the implementation of OCT in the EAE lab, especially with afoveate animals like rodents, remain undefined. We aimed to describe the dynamics of retinal injury in different mouse EAE models and outline the optimal experimental conditions, scan protocols, and analysis methods, comparing these to histology to confirm the pathological underpinnings.Methods: Using spectral-domain OCT, we analyzed the test-retest and the inter-rater reliability of volume, peripapillary, and combined horizontal and vertical line scans. We then monitored the thickness of the retinal layers in different EAE models: in wild-type (WT) C57Bl/6J mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG 35-55 ) or with bovine myelin basic protein (MBP), in TCR 2D2 mice immunized with MOG 35-55 , and in SJL/J mice immunized with myelin proteolipid lipoprotein ). Strain-matched control mice were shamimmunized. RGC density was counted on retinal flatmounts at the end of each experiment.Results: Volume scans centered on the optic disc showed the best reliability. Retinal changes during EAE were localized in the inner retinal layers (IRLs, the combination of the RNFL and the ganglion cell plus the inner plexiform layers). In WT, MOG 35-55 EAE, progressive thinning of IRL started rapidly after EAE onset, with 1/3 of total loss occurring during the initial 2 months. IRL thinning was associated with the degree of RGC loss and the severity of EAE. Sham-immunized SJL/J mice showed progressive IRL atrophy, which was accentuated in PLP-immunized mice. MOG 35-55 -immunized TCR 2D2 mice showed severe EAE and retinal thinning. MBP immunization led to very mild disease without significant retinopathy.(Continued on next page)

Sumoylation is a multi‐step protein modification reaction in which SUMO (small ubiquitin‐like modifier) proteins are covalently attached to lysine residues of substrate proteins. Here, we compare the sequences and structures of modifiers and enzymes involved in sumoylation with those of the related ubiquitination and neddylation cascades. By using available structural data on modifier/enzyme/substrate interactions, we discuss and model sumoylation complexes that include SUMO‐1 and the E1 and E2 enzymes Aos1‐uba2 and ubc9, or SUMO‐1 and E2 together with the E3 ligase RanBP2 and its substrate RanGAP1. Their comparison provides insight into the protein interactions underlying sumoylation, and suggests how SUMO proteins may be translocated between enzymes during the various steps of the protein modification reaction.