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.

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.

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.

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 gene inactivation weakens ESC pluripotency, and we identify SUMO2 modification of SATB2 by the E3 ligase ZFP451 as a potential driver of ESC differentiation.

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.

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.

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.

Chemotherapy followed by anti-CD137 mAb immunotherapy improves disease control in a mouse myeloma model.

Immunotherapy holds promise for multiple myeloma (MM) patients but little is known about how MM-induced immunosuppression influences response to therapy. Here, we investigated the impact of disease progression on immunotherapy efficacy in the Vk*MYC mouse model. Treatment with agonistic anti-CD137 (4-1BB) mAbs efficiently protected mice when administered early but failed to contain MM growth when delayed more than three weeks after Vk*MYC tumor cell challenge.

How DNA vicinity controls SUMO E3 ligase activity.

Little is known about the regulation of SUMO E3 ligases and how they are activated upon stress. New findings from the Reverter and Torres‐Rosell laboratories in demonstrate that vicinity of preferentially ssDNA activates the SUMO E3 ligase Nse2 when in complex with SMC5‐6.

SUMO conjugation - a mechanistic view.

The regulation of protein fate by modification with the small ubiquitin-related modifier (SUMO) plays an essential and crucial role in most cellular pathways. Sumoylation is highly dynamic due to the opposing activities of SUMO conjugation and SUMO deconjugation. SUMO conjugation is performed by the hierarchical action of E1, E2 and E3 enzymes, while its deconjugation involves SUMO-specific proteases.

A Fluorescent In Vitro Assay to Investigate Paralog-Specific SUMO Conjugation.

Protein modification with the small ubiquitin-related modifier SUMO is a potent regulatory mechanism implicated in a variety of biological pathways. In vitro sumoylation reactions have emerged as a versatile tool to identify and characterize novel SUMO enzymes as well as their substrates. Here, we present detailed protocols for the purification and fluorescent labeling of mammalian SUMO paralogs for their application in sumoylation assays.

The SUMO2/3 specific E3 ligase ZNF451-1 regulates PML stability.

The small ubiquitin related modifier SUMO regulates protein functions to maintain cell homeostasis. SUMO attachment is executed by the hierarchical action of E1, E2 and E3 enzymes of which E3 ligases ensure substrate specificity. We recently identified the ZNF451 family as novel class of SUMO2/3 specific E3 ligases and characterized their function in SUMO chain formation.

Structural basis for catalytic activation by the human ZNF451 SUMO E3 ligase.

E3 protein ligases enhance transfer of ubiquitin-like (Ubl) proteins from E2 conjugating enzymes to substrates by stabilizing the thioester-charged E2~Ubl in a closed configuration optimally aligned for nucleophilic attack. Here, we report biochemical and structural data that define the N-terminal domain of the Homo sapiens ZNF451 as the catalytic module for SUMO E3 ligase activity. The ZNF451 catalytic module contains tandem SUMO-interaction motifs (SIMs) bridged by a Pro-Leu-Arg-Pro (PLRP) motif.

A new vertebrate SUMO enzyme family reveals insights into SUMO-chain assembly.

SUMO chains act as stress-induced degradation tags or repair factor-recruiting signals at DNA lesions. Although E1 activating, E2 conjugating and E3 ligating enzymes efficiently assemble SUMO chains, specific chain-elongation mechanisms are unknown. E4 elongases are specialized E3 ligases that extend a chain but are inefficient in the initial conjugation of the modifier.

SUMO rules: regulatory concepts and their implication in neurologic functions.

Posttranslational modification of proteins by the small ubiquitin-like modifier (SUMO) is a potent regulator of various cellular events. Hundreds of substrates have been identified, many of them involved in vital processes like transcriptional regulation, signal transduction, protein degradation, cell cycle regulation, DNA repair, chromatin organization, and nuclear transport. In recent years, protein sumoylation increasingly attracted attention, as it could be linked to heart failure, cancer, and neurodegeneration.

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).

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.

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.

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.

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(+/-))).