TCR-independent CD137 (4-1BB) signaling promotes CD8+-exhausted T cell proliferation and terminal differentiation.

CD137 (4-1BB)-activating receptor represents a promising cancer immunotherapeutic target. Yet, the cellular program driven by CD137 and its role in cancer immune surveillance remain unresolved. Using T cell-specific deletion and agonist antibodies, we found that CD137 modulates tumor infiltration of CD8+-exhausted T (Tex) cells expressing PD1, Lag-3, and Tim-3 inhibitory receptors. T cell-intrinsic, TCR-independent CD137 signaling stimulated the proliferation and the terminal differentiation of Tex precursor cells through a mechanism involving the RelA and cRel canonical NF-κB subunits and Tox-dependent chromatin remodeling. While Tex cell accumulation induced by prophylactic CD137 agonists favored tumor growth, anti-PD1 efficacy was improved with subsequent CD137 stimulation in pre-clinical mouse models. Better understanding of T cell exhaustion has crucial implications for the treatment of cancer and infectious diseases. Our results identify CD137 as a critical regulator of Tex cell expansion and differentiation that holds potential for broad therapeutic applications.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy.

CD8+ T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ T cell function and limits the efficacy of cancer immunotherapy.

Stress-induced nuclear condensation of NELF drives transcriptional downregulation.

In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.

ZFP451-mediated SUMOylation of SATB2 drives embryonic stem cell differentiation.

The establishment of cell fates involves alterations of transcription factor repertoires and repurposing of transcription factors by post-translational modifications. In embryonic stem cells (ESCs), the chromatin organizers SATB2 and SATB1 balance pluripotency and differentiation by activating and repressing pluripotency genes, respectively. Here, we show that conditional Satb2 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation. Mutations of two SUMO-acceptor lysines of Satb2 (Satb2K →R ) or knockout of Zfp451 impair the ability of ESCs to silence pluripotency genes and activate differentiation-associated genes in response to retinoic acid (RA) treatment. Notably, the forced expression of a SUMO2-SATB2 fusion protein in either Satb2K →R or Zfp451-/- ESCs rescues, in part, their impaired differentiation potential and enhances the down-regulation of Nanog The differentiation defect of Satb2K →R ESCs correlates with altered higher-order chromatin interactions relative to Satb2wt ESCs. Upon RA treatment of Satb2wt ESCs, SATB2 interacts with ZFP451 and the LSD1/CoREST complex and gains binding at differentiation genes, which is not observed in RA-treated Satb2K →R cells. Thus, SATB2 SUMOylation may contribute to the rewiring of transcriptional networks and the chromatin interactome of ESCs in the transition of pluripotency to differentiation.

Ubc9 sumoylation controls SUMO chain formation and meiotic synapsis in Saccharomyces cerevisiae.

Posttranslational modification with the small ubiquitin-related modifier SUMO depends on the sequential activities of E1, E2, and E3 enzymes. While regulation by E3 ligases and SUMO proteases is well understood, current knowledge of E2 regulation is very limited. Here, we describe modification of the budding yeast E2 enzyme Ubc9 by sumoylation (Ubc9(*)SUMO). Although less than 1% of Ubc9 is sumoylated at Lys153 at steady state, a sumoylation-deficient mutant showed significantly reduced meiotic SUMO conjugates and abrogates synaptonemal complex formation. Biochemical analysis revealed that Ubc9(*)SUMO is severely impaired in its classical activity but promoted SUMO chain assembly in the presence of Ubc9. Ubc9(*)SUMO cooperates with charged Ubc9 (Ubc9~SUMO) by noncovalent backside SUMO binding and by positioning the donor SUMO for optimal transfer. Thus, sumoylation of Ubc9 converts an active enzyme into a cofactor and reveals a mechanism for E2 regulation that orchestrates catalytic (Ubc9~SUMO) and noncatalytic (Ubc9(*)SUMO) functions of Ubc9.

Ubc9 sumoylation regulates SUMO target discrimination.

Posttranslational modification with small ubiquitin-related modifier, SUMO, is a widespread mechanism for rapid and reversible changes in protein function. Considering the large number of known targets, the number of enzymes involved in modification seems surprisingly low: a single E1, a single E2, and a few distinct E3 ligases. Here we show that autosumoylation of the mammalian E2-conjugating enzyme Ubc9 at Lys14 regulates target discrimination. While not altering its activity toward HDAC4, E2-25K, PML, or TDG, sumoylation of Ubc9 impairs its activity on RanGAP1 and strongly activates sumoylation of the transcriptional regulator Sp100. Enhancement depends on a SUMO-interacting motif (SIM) in Sp100 that creates an additional interface with the SUMO conjugated to the E2, a mechanism distinct from Ubc9 approximately SUMO thioester recruitment. The crystal structure of sumoylated Ubc9 demonstrates how the newly created binding interface can provide a gain in affinity otherwise provided by E3 ligases.

Nedd4-2-dependent regulation of astrocytic Kir4.1 and Connexin43 controls neuronal network activity.

Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.

SUMO-activated target traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome.

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2, and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 6500 SUMO2/3 target proteins. The limited number of SUMO E3s provides the unique opportunity to systematically study E3 substrate wiring. We developed SUMO-activated target traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL (ZNF451-3), and ZMIZ2. SATTs enabled us to identify 427 SUMO1 and 961 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool to browse the dataset in an interactive manner. Overall, we uncover E3-to-target wiring of 1388 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

The Road Less Taken: Less Appreciated Pathways for Manipulating CD8+ T Cell Exhaustion.

Exhausted CD8+ T (Tex) cells are a distinct cell population that arise during persistent antigen exposure in the context of chronic infections and cancers. Although characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression and distinct transcriptional and epigenetic programs, Tex cells are heterogeneous. Among these, a self-renewing TCF-1+ Tex population, having unique characteristics and the ability to respond to immune-checkpoint blockade, gives rise to TCF-1- terminally Tex cells. These TCF-1+ cells have stem cell-like properties similar to memory T cell populations, but the signals that regulate the developmental pathways and relationships among exhausted cell populations are still unclear. Here, we review our current understanding of Tex cell biology, and discuss some less appreciated molecules and pathways affecting T cell exhaustion. We highlight two co-stimulatory receptors, CD226 and CD137, and their role in inducing or restraining T cell exhaustion, as well as signaling pathways that may be amenable to pharmacological inhibition with a focus on Phosphoinositide-3 Kinase and IL-2 partial agonists. Finally, we discuss novel methods that may increase TCF-1+ populations and therefore improve immunotherapy responsiveness. Understanding features of and pathways to exhaustion has important implications for the success of immunotherapy, including checkpoint blockade and adoptive T-cell transfer therapies.

Sumo control.

Sumoylation, the covalent attachment of SUMO peptide to cellular proteins, is an essential regulator of protein function involved in a wide range of cellular events. Deregulation of the SUMO pathway is implicated in the pathogenesis of several diseases, so it is important to understand how this system is controlled. Sumoylation is a highly dynamic regulatory mechanism, involving an energy dependent enzyme cascade for conjugation and another set of enzymes for deconjugation. In this chapter we will highlight the different mechanisms controlling the SUMO system.

Generation of a highly inducible Gal4-->Fluc universal reporter mouse for in vivo bioluminescence imaging.

Full understanding of the functional complexity of the protein interactome requires mapping of biomolecular complexes within the cellular environment over biologically relevant time scales. New approaches to imaging interacting protein partners in vivo will allow the study of functional proteomics of human biology and disease within the context of living animals. Herein, we describe a universal transgenic reporter mouse strain that expresses firefly luciferase (Fluc) under the regulatory control of a concatenated Gal4 promoter (Tg(G4F(+/-))). Using an adenovirus to deliver a fused binding-domain-activator chimera (Gal4BD-VP16), induction of bioluminescence in Tg(G4F(+/-)) tissues of up to 4 orders of magnitude was observed in fibroblasts, liver, respiratory epithelia, muscle, and brain. The Tg(G4F(+/-)) reporter strain allowed noninvasive detection of viral infectivity, duration of the infection as well as viral clearance in various tissues in vivo. To demonstrate protein-protein interactions in live mice, the well characterized interaction between tumor suppressor p53 (fused to Gal4BD) and large T antigen (TAg) (fused to VP16) was visualized in vivo by using a two-hybrid strategy. Hepatocytes of Tg(G4F(+/-)) mice transfected with p53/TAg demonstrated 48-fold greater induction of Fluc expression in vivo than noninteracting pairs. Furthermore, to demonstrate the feasibility of monitoring experimental therapy with siRNA in vivo, targeted knockdown of p53 resulted in markedly reduced light output, whereas use of a control siRNA had no effect on protein interaction-dependent induction of Fluc. Thus, this highly inducible Gal4-->Fluc conditional reporter strain should facilitate imaging studies of protein interactions, signaling cascades, viral dissemination, and therapy within the physiological context of the whole animal.

A second E2 for nedd8ylation expands substrate selection.

The ubiquitin-like modifier NEDD8 promotes substrate ubiquitination via its covalent linkage to ubiquitin cullin-RING ligases. This depends on a NEDD8-specific sequential enzymatic cascade of E1, E2, and E3 enzymes. The identification of a second E2 enzyme for nedd8ylation recently reported in Molecular Cell (Huang et al., 2009) reveals novel insights into the molecular mechanism of target cullin selection.

SUMO modification of the ubiquitin-conjugating enzyme E2-25K.

Post-translational modification with small ubiquitin-related modifier (SUMO) alters the function of many proteins, but the molecular mechanisms and consequences of this modification are still poorly defined. During a screen for novel SUMO1 targets, we identified the ubiquitin-conjugating enzyme E2-25K (Hip2). SUMO attachment severely impairs E2-25K ubiquitin thioester and unanchored ubiquitin chain formation in vitro. Crystal structures of E2-25K(1-155) and of the E2-25K(1-155)-SUMO conjugate (E2-25K(*)SUMO) indicate that SUMO attachment interferes with E1 interaction through its location on the N-terminal helix. The SUMO acceptor site in E2-25K, Lys14, does not conform to the consensus site found in most SUMO targets (PsiKXE), and functions only in the context of an alpha-helix. In contrast, adjacent SUMO consensus sites are modified only when in unstructured peptides. The demonstration that secondary structure elements are part of SUMO attachment signals could contribute to a better prediction of SUMO targets.

The RanBP2 SUMO E3 ligase is neither HECT- nor RING-type.

Post-translational modification with the ubiquitin-related protein SUMO1 requires the E1 enzyme Aos1-Uba2 and the E2 enzyme Ubc9. Distinct E3 ligases strongly enhance modification of specific targets. The SUMO E3 ligase RanBP2 (also known as Nup358) has no obvious similarity to RING- or HECT-type enzymes. Here we show that RanBP2's 30-kDa catalytic fragment is a largely unstructured protein. Despite two distinct but partially overlapping 79-residue catalytic domains, one of which is sufficient for maximal activity, RanBP2 binds to Ubc9 in a 1:1 stoichiometry. The identification of nine RanBP2 and three Ubc9 side chains that are important for RanBP2-dependent SUMOylation indicates largely hydrophobic interactions. These properties distinguish RanBP2 from all other known E3 ligases, and we speculate that RanBP2 exerts its catalytic effect by altering Ubc9's properties rather than by mediating target interactions.

Preparation of sumoylated substrates for biochemical analysis.

Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

SUMO: ligases, isopeptidases and nuclear pores.

Small ubiquitin-related modifier (SUMO) proteins are reversibly coupled to numerous intracellular targets and modulate their interactions, localization, activity or stability. Recent advances in the SUMO field have uncovered the first SUMO E3 ligases and point to a complex family of isopeptidases. SUMO has been linked to many different pathways, including nucleocytoplasmic transport. Modifying enzymes and an isopeptidase have been detected at nuclear pore complexes. In addition, studies in yeast suggest a requirement of SUMO conjugation for nuclear protein import, and specific SUMO targets depend on modification for nuclear import or export.

Analysis of Sumoylation.

Protein regulation by reversible attachment of SUMO (small ubiquitin-related modifier) plays an important role in several cellular processes such as transcriptional regulation, nucleo-cytoplasmic transport, cell-cycle progression, meiosis, and DNA repair. However, most sumoylated proteins are of marginal abundance at steady state levels, which is due to strict regulation and/or rapid turnover of modification and de-modification. Consequently, analysis of protein sumoylation in vivo is very challenging. Nonetheless, a novel method was established that allows detection of sumoylated proteins at endogenous levels from vertebrate cells and tissues. This approach involves the enrichment of sumoylated proteins by immunoprecipitation followed by peptide elution. After endogenous substrate sumoylation is verified, addressing its functional consequences is the next logical step. This requires SUMO site mapping that benefits from larger quantities of modified protein. Here, we shortly describe strategies to achieve efficient in vitro sumoylation of many substrates.

Biochemical characterization of SUMO-conjugating enzymes by in vitro sumoylation assays.

The small ubiquitin-related modifier (SUMO) is a protein of ~10kDa that is covalently conjugated to its substrate proteins in an enzymatic process called sumoylation. This posttranslational modification is an essential regulatory mechanism that plays crucial roles in many cellular pathways. It allows rapid adaptation to environmental changes by switching protein functions due to alternate complex assemblies, changes in intracellular localization, enzymatic activity, or stability. SUMO conjugation is executed by the hierarchical action of E1, E2, and E3 enzymes. Both E2 and E3 enzymes contribute to substrate specificity but with E3 ligases being the more important for this. E1 and E2 activities are essential for all sumoylation reactions but usually-with a few exceptions-modify substrates only inefficiently. Hence, most substrates require the additional action of an E3 ligase or a cofactor. Here, we describe methods to distinguish a bona fide E3 ligase from a cofactor activity by using in vitro sumoylation assays.