At the crossroads of ubiquitin signaling and mass spectrometry

Ubiquitin signaling regulates a wide variety of cellular events, although it is mostly known to mediate protein degradation by the proteasome complex. The rapid development in mass spectrometry offers state-of-the-art technologies for addressing biological challenges in ubiquitin signaling. The First Conference on Proteomics of Protein Degradation & Ubiquitin Pathways in Vancouver, Canada, covers the latest progress in key topics of the field and fosters collaborative interactions among researchers.     

Proteomic Analysis of Rat Hypothalamus Revealed the Role of Ubiquitin–Proteasome System in the Genesis of DR or DIO

Obesity has become a global epidemic, contributing to the increasing burdens of cardiovascular disease and type 2 diabetes. However, the precise molecular mechanisms of obesity remain poorly elucidated. The hypothalamus plays a major part in regulating energy homeostasis by integrating all kinds of nutritional signals. This study investigated the hypothalamus protein profile in diet-induced obese (DIO) and diet-resistant (DR) rats using two dimensional gel electrophoresis (2-DE) combined with MALDI-TOF/TOF–MS analysis. Twenty-two proteins were identified in the hypothalamus of DIO or DR rats. These include metabolic enzymes, antioxidant proteins, proteasome related proteins, and signaling proteins, some of which are related to AMP-activated protein kinase (AMPK) signaling or mitochondrial respiration. Among these proteins, in comparison with the normal-diet group, Ubiquitin was significantly decreased in DR rats but not changed in DIO rats, while Ubiquitin carboxyl-terminal esterase L1 (UCHL-1) was decreased in DIO rats but not changed in DR rats. The expression level of Ubiquitin and UCHL-1 were further validated using Western blot analysis. Our study reveals that Ubiquitin and UCHL-1 are obesity-related factors in the hypothalamus that may play an important role in the genesis of DR or DIO by interfering with the integrated signaling network that control energy balance and feeding.     

IAPS and ubiquitylation

The Inhibitor of apoptosis (IAP) proteins are key negative regulators of cell death, whose amplification has been correlated with tumor progression. Due to the presence of a RING domain, IAP proteins are classed as ubiquitin ligases and regulate cell survival by orchestrating a variety of ubiquitin modifications. Ubiquitin protein modification is fundamental in cell signaling and different ubiquitin modifications may label proteins for destruction, relocalization or provide a recruitment platform for ubiquitin binding proteins. Ubiquitin performs a myriad of different functions because it can be conjugated to a large range of target proteins through numerous different types of ubiquitin linkages. Despite the fact that ubiquitin is extremely versatile, the E3s such as the IAPs provide an important level of control due to their specificity for certain substrates. Several recent reviews have discussed the role of IAPs in regulating immune signaling so we have therefore focused our review on the interplay between IAPs and ubiquitin and discussed the importance of this relationship for the regulation of themselves, specific substrates and various cell death and survival signaling pathways.     

FIH-1, a Novel Interactor of Mindbomb,Functions as an Essential Anti-Angiogenic Factor during Zebrafish Vascular Development

Objective

It has been shown that Mindbomb (Mib), an E3 Ubiquitin ligase, is an essential modulator of Notch signaling during development. However, its effects on vascular development remain largely unknown.

Approaches and Results

We identified a number of novel proteins that physically interact with Mib, including the Factor Inhibiting Hypoxia Inducible Factor 1 (FIH-1, also known as HIF1AN) from a yeast two hybrid screen, as previously reported. In cultured cells, FIH-1 colocalizes with Mib1, corroborating their potential interaction. In zebrafish embryos, FIH-1 appears to modulate VEGF-A signaling activity; depletion of fih-1 induces ectopic expression of vascular endothelial growth factor–a (vegfa) and leads to exuberant ectopic sprouts from intersegmental vessels (ISVs). Conversely, over-expression of fih-1 substantially attenuates the formation of ISVs, which can be rescued by concurrent over-expression of vegfa, indicating that FIH-1/HIF1AN may fine tune VEGF-A signaling.

Conclusions

Taken together, our data suggest that FIH-1 interacts with Mib E3 Ubiquitin ligase and modulates vascular development by attenuating VEGF-A signaling activity.     

RNF4—A Paradigm for SUMOylation‐Mediated Ubiquitination

Covalent modifications by Small Ubiquitin‐like MOdifier (SUMO) and ubiquitin conjugation are now recognized as independent posttranslational modifications (PTMs) employed by cells to reversibly regulate cellular signaling. SUMOylation in particular has emerged as a crucial cellular mechanism involved in multiple pathologies, including cancers, cardiovascular diseases, immunological and neurological disorders, as well as aging. Convergence of these two PTMs result in the ubiquitination of SUMOylated proteins, adding complexity in the modulation of protein functions. The SUMO‐Targeted Ubiquitin Ligases (STUbL) mediate this process, and RNF4, the mammalian STUbL, has been at the forefront in the understanding of this phenomenon. It has been shown to play important roles in a variety of cellular events, ranging from the maintenance of genomic integrity and hence, oncogenesis, to a role in development. Recent identification of direct and indirect RNF4 targets has revealed that the SUMOylation machinery is in itself targeted by RNF4, highlighting the complex nature of the signaling circuitry tightly regulating these processes. This review will touch upon both SUMOylation and ubiquitination, and will focus on how RNF4, which is at the heart of both these PTMs, modulates cellular signaling and promotes protein degradation. Moreover, the potential of therapeutically targeting RNF4 to improve cancer treatment is also explored.     

The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like β-grasp domains

Background  

Ubiquitin (Ub)-mediated signaling is one of the hallmarks of all eukaryotes. Prokaryotic homologs of Ub (ThiS and MoaD) and E1 ligases have been studied in relation to sulfur incorporation reactions in thiamine and molybdenum/tungsten cofactor biosynthesis. However, there is no evidence for entire protein modification systems with Ub-like proteins and deconjugation by deubiquitinating enzymes in prokaryotes. Hence, the evolutionary assembly of the eukaryotic Ub-signaling apparatus remains unclear.     

The novel functions of ubiquitination in signaling

Ubiquitin is best known for its function in targeting proteins for degradation by the proteasome. Recent studies have revealed several new functions of ubiquitin that are independent of proteasomal degradation. These functions include the novel signaling roles of ubiquitin in DNA repair and the activation of protein kinases such as IkappaB kinase. In both cases, a novel form of polyubiquitin chain linked through lysine-63 of ubiquitin plays an important regulatory role. Monoubiquitination also has signaling roles that are distinct from those of polyubiquitination, as illustrated from the studies of DNA repair. Thus, polyubiquitination and monoubiquitination have emerged as important signaling mechanisms that control diverse physiological and pathological processes.     

Drosophila Epsin's role in Notch ligand cells requires three Epsin protein functions: the lipid binding function of the ENTH domain, a single Ubiquitin interaction motif, and a subset of the C-terminal protein binding modules

Epsin is an endocytic protein that binds Clathrin, the plasma membrane, Ubiquitin, and also a variety of other endocytic proteins through well-characterized motifs. Although Epsin is a general endocytic factor, genetic analysis in Drosophila and mice revealed that Epsin is essential specifically for internalization of ubiquitinated transmembrane ligands of the Notch receptor, a process required for Notch activation. Epsin's mechanism of function is complex and context-dependent. Consequently, how Epsin promotes ligand endocytosis and thus Notch signaling is unclear, as is why Notch signaling is uniquely dependent on Epsin. Here, by generating Drosophila lines containing transgenes that express a variety of different Epsin deletion and substitution variants, we tested each of the five protein or lipid interaction modules for a role in Notch activation by each of the two ligands, Serrate and Delta. There are five main results of this work that impact present thinking about the role of Epsin in ligand cells. First, we discovered that deletion or mutation of both UIMs destroyed Epsin's function in Notch signaling and had a greater negative impact on Epsin activity than removal of any other module type. Second, only one of Epsin's two UIMs was essential. Third, the lipid-binding function of the ENTH domain was required only for maximal Epsin activity. Fourth, although the C-terminal Epsin modules that interact with Clathrin, the adapter protein complex AP-2, or endocytic accessory proteins were necessary collectively for Epsin activity, their functions were highly redundant; most unexpected was the finding that Epsin's Clathrin binding motifs were dispensable. Finally, we found that signaling from either ligand, Serrate or Delta, required the same Epsin modules. All of these observations are consistent with a model where Epsin's essential function in ligand cells is to link ubiquitinated Notch ligands to Clathrin-coated vesicles through other Clathrin adapter proteins. We propose that Epsin's specificity for Notch signaling simply reflects its unique ability to interact with the plasma membrane, Ubiquitin, and proteins that bind Clathrin.     

Differential modification of Ras proteins by ubiquitination

Ras proteins are essential components of signal transduction pathways that control cell proliferation, differentiation, and survival. It is well recognized that the functional versatility of Ras proteins is accomplished through their differential compartmentalization, but the mechanisms that control their spatial segregation are not fully understood. Here we show that HRas is subject to ubiquitin conjugation, whereas KRas is refractory to this modification. The membrane-anchoring domain of HRas is necessary and sufficient to direct the mono- and diubiquitination of HRas. Ubiquitin attachment to HRas stabilizes its association with endosomes and modulates its ability to activate the Raf/MAPK signaling pathway. Therefore, differential ubiquitination of Ras proteins may control their location-specific signaling activities.     

Counterbalance of ligand‐ and self‐coupled motions characterizes multispecificity of ubiquitin

Date hub proteins are a type of proteins that show multispecificity in a time‐dependent manner. To understand dynamic aspects of such multispecificity we studied Ubiquitin as a typical example of a date hub protein. Here we analyzed 9 biologically relevant Ubiquitin‐protein (ligand) heterodimer structures by using normal mode analysis based on an elastic network model. Our result showed that the self‐coupled motion of Ubiquitin in the complex, rather than its ligand‐coupled motion, is similar to the motion of Ubiquitin in the unbound condition. The ligand‐coupled motions are correlated to the conformational change between the unbound and bound conditions of Ubiquitin. Moreover, ligand‐coupled motions favor the formation of the bound states, due to its in‐phase movements of the contacting atoms at the interface. The self‐coupled motions at the interface indicated loss of conformational entropy due to binding. Therefore, such motions disfavor the formation of the bound state. We observed that the ligand‐coupled motions are embedded in the motions of unbound Ubiquitin. In conclusion, multispecificity of Ubiquitin can be characterized by an intricate balance of the ligand‐ and self‐coupled motions, both of which are embedded in the motions of the unbound form.     

蛋白质的泛素化修饰在细胞应激反应中的作用

泛素是真核细胞内广泛存在的一种高度保守的蛋白质。在特定泛素化酶催化下实现的蛋白质泛素化修饰反应能够高选择性地降解细胞中的特定信号蛋白质,对维持细胞正常的生理功能具有非常重要的作用。另外,某些泛素化修饰反应也能够实现与蛋白质降解无关的功能调控作用。p53、NF-κB和GADD45α是在细胞应激损伤反应中具有广泛调控作用的信号蛋白,发生在这些分子上的泛素化修饰反应是它们发挥相关分子机制的重要基础。     

泛素特异蛋白酶4研究进展

泛素特异蛋白酶4(ubiquitin-specific protease 4,USP4)是一种重要的去泛素化酶,它通过识别特异性靶蛋白,使之去泛素化来阻碍其降解或改变其特性,在肿瘤、病毒感染及多种信号通路中发挥重要调节作用。但目前有关USP4在肿瘤形成和发展过程中究竟行使癌基因抑或是抑癌基因的功能尚存在争议。本文从USP4的结构、功能及信号通路等方面对其研究进展作一介绍。     

Ring Finger Protein RNF169 Antagonizes the Ubiquitin-dependent Signaling Cascade at Sites of DNA Damage

Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance.     

USP33, a new player in lung cancer,mediates Slit-Robo signaling

Ubiquitin specific protease 33 (USP33) is a multifunctional protein regulating diverse cellular processes. The expression and role ofUSP33 in lung cancer remain unexplored. In this study, we show that USP33 is down-regulated in multiple cohorts of lung cancer patients and that low expression of USP33 is associated with poor prognosis. USP33 mediates Slit-Robo signaling in lung cancer cell migration. Downregulation of USP33 reduces the protein stability of Robo1inlungcancer cells, providing apreviouslyunknown mechanism for USP33 function in mediating Slit activity in lung cancer cells. Taken together, USP33 is a new player in lung cancer that regulates Slit-Robo signaling. Our data suggest that USP33 may be a candidate tumor suppressor for lung cancer with potential as a prognostic marker.     

Intrinsic Flexibility of Ubiquitin on Proliferating Cell Nuclear Antigen (PCNA) in Translesion Synthesis

Ubiquitin conjugation provides a crucial signaling role in hundreds of cellular pathways; however, a structural understanding of ubiquitinated substrates is lacking. One important substrate is monoubiquitinated PCNA (PCNA-Ub), which signals for recruitment of damage-tolerant polymerases in the translesion synthesis (TLS) pathway of DNA damage avoidance. We use a novel and efficient enzymatic method to produce PCNA-Ub at high yield with a native isopeptide bond and study its Usp1/UAF1-dependent deconjugation. In solution we find that the ubiquitin moiety is flexible relative to the PCNA, with its hydrophobic patch mostly accessible for recruitment of TLS polymerases, which promotes the interaction with polymerase η. The studies are a prototype for the nature of the ubiquitin modification.     

Ubiquitin-regulating effector proteins from Legionella

Ubiquitin is relatively modest in size but involves almost entire cellular signaling pathways. The primary role of ubiquitin is maintaining cellular protein homeostasis. Ubiquitination regulates the fate of target proteins using the proteasome- or autophagy-mediated degradation of ubiquitinated substrates, which can be either intracellular or foreign proteins from invading pathogens. Legionella, a gram-negative intracellular pathogen, hinders the host-ubiquitin system by translocating hundreds of effector proteins into the host cell’s cytoplasm. In this review, we describe the current understanding of ubiquitin machinery from Legionella. We summarize structural and biochemical differences between the host-ubiquitin system and ubiquitin-related effectors of Legionella. Some of these effectors act much like canonical host-ubiquitin machinery, whereas others have distinctive structures and accomplish non-canonical ubiquitination via novel biochemical mechanisms.     

Chemical shift assignments for <Emphasis Type="Italic">S. cerevisiae</Emphasis> Ubc13

The ubiquitination pathway controls several human cellular processes, most notably protein degradation. Ubiquitin, a small signaling protein, is activated by the E1 activating enzyme, transferred to an E2 conjugating enzyme, and then attached to a target substrate through a process that can be facilitated by an E3 ligase enzyme. The enzymatic mechanism of ubiquitin transfer from the E2 conjugating enzyme onto substrate is not clear. The highly conserved HPN motif in E2 catalytic domains is generally thought to help stabilize an oxyanion intermediate formed during ubiquitin transfer. However recent work suggests this motif is instead involved in a structural, non-enzymatic role. As a platform to better understand the E2 catalyzed ubiquitin transfer mechanism, we determined the chemical shift assignments of S. cerevisiae E2 enzyme Ubc13.