Negr1-Derived Peptides Trigger ALK Degradation and Halt Neuroblastoma Progression In Vitro and In Vivo.

Neuroblastoma is among the most common childhood cancers. Neuroblastoma in advanced stages is one of the most intractable pediatric cancers, notwithstanding the recent therapeutic advances. ALK mutations are among the leading cause of hereditary neuroblastoma and account for more than 14% of the somatically acquired alterations. ALK kinase activity is currently one of the main targets for pharmacological strategies. However, evidence from ALK fusion-positive lung cancer studies has shown that resistance to ALK inhibition arises during the therapy, causing a relapse within several years. IgLONs are membrane-bound proteins involved in cell-to-cell adhesion. The expression of the IgLON family results altered in different cancers. We found that the IgLON member Negr1 is downregulated in neuroblastoma. The ectopic overexpression of Negr1 impairs neuroblastoma growth in vitro and in vivo. Negr1 exists as a GPI-anchored membrane-bound protein and as a soluble protein released upon metalloprotease cleavage. We generated and characterized a panel of Negr1-derived peptides. The treatment with Negr1 protein and derived peptides induce ALK downregulation and halt neuroblastoma progression in vitro and in vivo.

Non-covalent Interaction With SUMO Enhances the Activity of Human Cytomegalovirus Protein IE1.

Viruses interact with the host cellular pathways to optimize cellular conditions for replication. The Human Cytomegalovirus (HCMV) Immediate-Early protein 1 (IE1) is the first viral protein to express during infection. It is a multifunctional and conditionally essential protein for HCMV infection. SUMO signaling regulates several cellular pathways that are also targets of IE1. Consequently, IE1 exploits SUMO signaling to regulate these pathways. The covalent interaction of IE1 and SUMO (IE1-SUMOylation) is well studied. However, the non-covalent interactions between SUMO and IE1 are unknown. We report two SUMO-Interacting Motifs (SIMs) in IE1, one at the end of the core domain and another in the C-terminal domain. NMR titrations showed that IE1-SIMs bind to SUMO1 but not SUMO2. Two critical functions of IE1 are inhibition of SUMOylation of Promyelocytic leukemia protein (PML) and transactivation of viral promoters. Although the non-covalent interaction of IE1 and SUMO is not involved in the inhibition of PML SUMOylation, it contributes to the transactivation activity. The transactivation activity of IE1 was previously correlated to its ability to inhibit PML SUMOylation. Our results suggest that transactivation and inhibition of PML SUMOylation are independent activities of IE1.

A Fluorescence-Based Assay to Monitor SUMOylation in Real-Time.

The small ubiquitin-like modifier (SUMO) is an important post-translational modifier that regulates various cellular processes. Extensive investigations have been made to comprehend the enzymatic process and consequence of SUMOylation. In vitro SUMOylation assays are invaluable for understanding the fundamental mechanisms of SUMOylation. A majority of these assays monitor changes in the size of the substrate upon SUMO conjugation. Current methods typically detect the size difference through SDS-PAGE and western blots, which makes these methods cumbersome, error-prone, and time-consuming. Here, we describe a fluorescence-based assay for real-time detection of SUMOylation. In the method, a fluorophore-tagged substrate is used in the SUMOylation reaction. Upon SUMOylation, the size and correlation time (τc ) of the substrate increases, and so does its anisotropy. The rate of change in anisotropy with time reflects the efficiency of the SUMOylation machinery. The real-time SUMOylation assay protocol is elegant, time-saving, and less prone to errors. © 2020 Wiley Periodicals LLC. Basic Protocol: Fluorescent anisotropy-based in vitro SUMOylation assay.

Monitoring protein ubiquitination and SUMOylation in real-time by NMR.

The covalent conjugation of ubiquitin (Ub), known as ubiquitination, is a multi-step reaction involving multiple enzymes. We report a real-time, tag-free method to monitor protein ubiquitination by NMR spectroscopy under physiological conditions. The approach is also applicable for monitoring other ubiquitin-like modifications, and the disassembly of Ub polymers.

The Viral SUMO-Targeted Ubiquitin Ligase ICP0 is Phosphorylated and Activated by Host Kinase Chk2.

When the herpes simplex virus (HSV) genome enters the nucleus for replication and transcription, phase-segregated nuclear protein bodies called Promyelocytic leukemia protein nuclear bodies (PML NBs) colocalize with the genome and repress it. HSV encodes a small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL) infected cell polypeptide 0 (ICP0) that degrades PML NBs to alleviate the repression. The molecular details of the mechanism used by ICP0 to target PML NBs are unclear. Here, we identify a bona fide SUMO-interacting motif in ICP0 (SIM-like sequence [SLS] 4) that is essential and sufficient to target SUMOylated proteins in PML NBs such as the PML and Sp100. We shown that phosphorylation of SLS4 creates new salt bridges between SUMO and SLS4, increases the SUMO/SLS4 affinity, and switches ICP0 into a potent STUbL. HSV activates the Ataxia-telangiectasia-mutated kinase-Checkpoint kinase 2 (ATM-Chk2) pathway to regulate the cell cycle of the host. We report that the activated Chk2 also phosphorylates ICP0 at SLS4 and enhances its STUbL activity. Our results uncover that a viral STUbL counters antiviral response by exploiting an unprecedented cross-talk of three post-translational modifications: ubiquitination, SUMOylation, and phosphorylation.

Casein kinase-2-mediated phosphorylation increases the SUMO-dependent activity of the cytomegalovirus transactivator IE2.

Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication. In particular, phosphorylation and SUMOylation can distinctly regulate the activity of the human cytomegalovirus (HCMV) transactivator immediate early 2 (IE2). However, the molecular mechanism of this process is unknown. Using various structural, biochemical, and cell-based approaches, here we uncovered that IE2 exploits a cross-talk between phosphorylation and SUMOylation. A scan for small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) revealed two SIMs in IE2, and a real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhances IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies disclosed that IE2 is a SUMO cis-E3 ligase. We also found that two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1 are phosphorylated in vitro and in cells. The phosphorylation drastically increased IE2-SUMO affinity, IE2 SUMOylation, and cis-E3 activity of IE2. Additional salt bridges between the phosphoserines and SUMO accounted for the increased IE2-SUMO affinity. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism whereby SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2 work in tandem to enable transcriptional regulation of viral gene.

A conserved and buried edge-to-face aromatic interaction in small ubiquitin-like modifier (SUMO) has a role in SUMO stability and function.

Aromatic amino acids buried at a protein's core are often involved in mutual paired interactions. Ab initio energy calculations have highlighted that the conformational orientations and the effects of substitutions are important for stable aromatic interactions among aromatic rings, but studies in the context of a protein's fold and function are elusive. Small ubiquitin-like modifier (SUMO) is a common post-translational modifier that affects diverse cellular processes. Here, we report that a highly conserved aromatic triad of three amino acids, Phe36-Tyr51-Phe64, is a unique SUMO signature that is absent in other ubiquitin-like homologous folds. We found that a specific edge-to-face conformation between the Tyr51-Phe64 pair of interacting aromatics is vital to the fold and stability of SUMO. Moreover, the noncovalent binding of SUMO-interacting motif (SIM) at the SUMO surface was critically dependent on the paired aromatic interactions buried at the core. NMR structural studies revealed that perturbation of the Tyr51-Phe64 conformation disrupts several long-range tertiary contacts in SUMO, leading to a heterogeneous and dynamic protein with attenuated SUMOylation both in vitro and in cells. A subtle perturbation of the edge-to-face conformation by a Tyr to Phe substitution significantly decreased stability, SUMO/SIM affinity, and the rate of SUMOylation. Our results highlight that absolute co-conservation of specific aromatic pairs inside the SUMO protein core has a role in its stability and function.

Structural and functional analysis of SMO-1, the SUMO homolog in Caenorhabditis elegans.

SUMO proteins are important post-translational modifiers involved in multiple cellular pathways in eukaryotes, especially during the different developmental stages in multicellular organisms. The nematode C. elegans is a well known model system for studying metazoan development and has a single SUMO homolog, SMO-1. Interestingly, SMO-1 modification is linked to embryogenesis and development in the nematode. However, high-resolution information about SMO-1 and the mechanism of its conjugation is lacking. In this work, we report the high-resolution three dimensional structure of SMO-1 solved by NMR spectroscopy. SMO-1 has flexible N-terminal and C-terminal tails on either side of a rigid beta-grasp folded core. While the sequence of SMO-1 is more similar to SUMO1, the electrostatic surface features of SMO-1 resemble more with SUMO2/3. SMO-1 can bind to typical SUMO Interacting Motifs (SIMs). SMO-1 can also conjugate to a typical SUMOylation consensus site as well as to its natural substrate HMR-1. Poly-SMO-1 chains were observed in-vitro even though SMO-1 lacks any consensus SUMOylation site. Typical deSUMOylation enzymes like Senp2 can cleave the poly-SMO-1 chains. Despite being a single gene, the SMO-1 structure allows it to function in a large repertoire of signaling pathways involving SUMO in C. elegans. Structural and functional features of SMO-1 studies described here will be useful to understand its role in development.