Evaluation of direct effects of protein ubiquitylation using computational analysis

Ubiquitylation is an important regulatory mechanism in the eukaryotic cell. A large volume of experimental data on protein ubiquitylation has been acquired in recent years. Particular ubiquitylated lysine residues were also identified. This allows us to analyze co-localization of ubiquitylation sites and functionally important protein domains, following the idea that ubiquitylation can directly affect protein functional activity. Computational analysis suggests that ubiquitylation can affect the functional activity of some proteins through direct steric effects. (1) Ubiquitylation can block protein functional domains/active site or cause accessibility limitations. It also (2) causes steric disturbances for homo-oligomerization and (3) influences heterologous protein interactions, impeding the binding of target protein with its partners. (4) Interaction with partner proteins can be disturbed by restricted conformational flexibility. Any of these effects will result in a decrease of target protein activity. Thus, we suggest a new “loss-of-function” mechanism of protein regulation by ubiquitylation.     

The regulation of necroptosis by ubiquitylation

Necroptosis is a programmed necrosis that is mediated by receptor-interacting protein kinases RIPK1, RIPK3 and the mixed lineage kinase domain-like protein, MLKL. Necroptosis must be strictly regulated to maintain normal tissue homeostasis, and dysregulation of necroptosis leads to the development of various inflammatory, infectious, and degenerative diseases. Ubiquitylation is a widespread post-translational modification that is essential for balancing numerous physiological processes. Over the past decade, considerable progress has been made in the understanding of the role of ubiquitylation in regulating necroptosis. Here, we will discuss the regulatory functions of ubiquitylation in necroptosis signaling pathway. An enhanced understanding of the ubiquitylation enzymes and regulatory proteins in necroptotic signaling pathway will be exploited for the development of new therapeutic strategies for necroptosis-related diseases.

     

蛋白质泛素化系统

泛素化是单个或多个泛素在泛素激活酶,泛素结合酶及泛素蛋白质连接酶的作用下共价修饰底物蛋白质的过程,近年来的研究发现,许多含环指的蛋白质本身是蛋白质泛素连接酶,或是多亚基连接酶中的重要成分。由于细胞内可表达200以上的环指蛋白,并且多亚基连接酶可利用同一环指蛋白但不同的底物识别蛋白。这些研究极大地丰富了对泛素化系统酶的认识,也使进一步调节和干预连接酶与底物的相互作用成为可能,新近的研究还发现,泛素化不仅可导致蛋白质的降解,还可直接影响蛋白质的活性和细胞内定位,是调节细胞内蛋白质功能和水平的主要机制之一。     

Identification of developmentally expressed proteins that functionally interact with Nedd4 ubiquitin ligase.

Nedd4 is a HECT domain-containing ubiquitin ligase that mediates ubiquitylation and proteasome degradation of target proteins. The molecular basis for the interaction of Nedd4 with substrates lies in its WW domains, which can bind proline-rich (PY) domains in target proteins. Nedd4 is a developmentally expressed protein and may have a fundamental role to play in embryonic processes. However, whether Nedd4 has such a function is currently unknown, in part because few developmentally regulated ubiquitylation substrates have been identified or characterized. We have carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation embryo that are able to interact with Nedd4. Characterization of their functional interaction with Nedd4 in vitro and in vivo demonstrated that three of the four are bona fide Nedd4 binding partners, and two have the capacity to be ubiquitylation substrates. One of these is the first identified nonviral substrate for Nedd4-mediated monoubiquitylation. Interestingly, neither of these two ubiquitylated proteins interacts with Nedd4 through PY-mediated mechanisms. For one of the three Nedd4 binding partners, there was no discernable evidence of ubiquitylation. However, this protein clearly associates with Nedd4 through its PY domains and can alter the location of Nedd4 in cells, suggesting a role other than as a ubiquitylation substrate.     

MultiDsk: A Ubiquitin-Specific Affinity Resin

Ubiquitylation is a highly diverse and complex post-translational modification for the regulation of protein function and stability. Studies of ubiquitylation have, however, been hampered by its rapid reversal in cell extracts, for example through the action of de-ubiquitylating enzymes (DUBs). Here we describe a novel ubiquitin-binding protein reagent, MultiDsk, composed of an array of five UBA domains from the yeast ubiquitin-binding protein Dsk2, fused to GST. MultiDsk binds ubiquitylated substrates with unprecedented avidity, and can be used as both an affinity resin to study protein ubiquitylation, and to effectively protect ubiquitylated proteins from the action of DUBs and the proteasome in crude cell extracts. We use the resin to show that the Def1 protein becomes ubiquitylated in response to DNA damage, and to isolate ubiquitylated forms of RNA polymerase II.     

泛素蛋白磷酸化修饰的功能与解析

泛素化是存在于真核生物中一种重要的翻译后修饰过程,参与调控包括蛋白质降解在内的多种生命活动。实现这一调控过程需要将一个由76个氨基酸组成的泛素蛋白共价连接到底物蛋白上。同时,泛素本身也存在多种翻译后修饰,包括泛素化、磷酸化、乙酰化等,进一步丰富了泛素的修饰类型,决定了底物蛋白不同的命运。近年来,伴随着第65位丝氨酸磷酸化泛素蛋白参与调控线粒体自噬这一突破性进展,泛素蛋白其余磷酸化位点的功能研究也获得越来越多的关注。本文根据目前已有的国内外研究和报道,总结了泛素蛋白已知的磷酸化修饰位点,梳理了泛素蛋白第12位和66位苏氨酸、第57位和65位丝氨酸等位点的磷酸化修饰对其生物物理特性带来的改变,并对相应修饰位点所涉及的生物学功能调控进行了综述。     

组蛋白泛素化修饰及其在DNA损伤应答中的作用

泛素化修饰是真核生物细胞内重要的翻译后修饰类型,通过调节蛋白质活性、稳定性和亚细胞定位广泛参与细胞内各项信号传导与代谢过程,对维持正常生命活动具有重要意义。组蛋白作为染色质中主要的蛋白成分,与DNA复制转录、修复等行为密切相关,是研究翻译后修饰的热点。DNA损伤后,组蛋白泛素化修饰通过调节核小体结构、激活细胞周期检查点、影响修复因子的招募与装配等诸多途径参与损伤应答。同时,组蛋白泛素化修饰还能调节其他位点翻译后修饰,并通过这种串扰(crosstalk)作用调节DNA损伤应答。本文介绍了组蛋白泛素化修饰的主要位点和相关组分(包括E3连接酶、去泛素化酶与效应分子),以及这些修饰作用共同编译形成的信号网络在DNA损伤应答中的作用,最后总结了目前该领域研究所面临的一些问题,以期为科研人员进一步探索组蛋白密码在DNA损伤应答中的作用提供参考。     

Ubiquitylation of the transducin betagamma subunit complex. Regulation by phosducin

G proteins (Galphabetagamma) are essential signaling molecules, which dissociate into Galpha and Gbetagamma upon activation by heptahelical membrane receptors. We have identified the betagamma subunit complex of the photoreceptor-specific G protein, transducin (T), as a target of the ubiquitin-proteasome pathway. Ubiquitylated species of the transducin gamma-subunit (Tgamma) but not the alpha- or beta-subunits were assembled de novo in bovine photoreceptor preparations. In addition, Tgamma was exclusively ubiquitylated when Tbetagamma was dissociated from Talpha. Ubiquitylation of Tbetagamma on Tgamma was selectively catalyzed by human ubiquitin-conjugating enzymes UbcH5 and UbcH7 and was coincident with degradation of the entire Tbetagamma subunit complex in vitro by a mechanism requiring ATP and the proteasome. We also show that Tbetagamma association with phosducin, a photoreceptor-specific protein of unknown physiological function, blocks Tbetagamma ubiquitylation and subsequent degradation. Phosphorylation of phosducin by Ca(2+)/calmodulin-dependent protein kinase II, which inhibits phosducin-Tbetagamma complex formation, completely restored Tbetagamma ubiquitylation and degradation. We conclude that Tbetagamma is a substrate of the ubiquitin-proteasome pathway and suggest that phosducin serves to protect Tbetagamma following the light-dependent dissociation of Talphabetagamma.     

Ubiquitylation is required for degradation of transmembrane surface proteins in trypanosomes

The surface of Trypanosoma brucei is dominated by glycosyl-phosphatidylinositol (GPI)-anchored proteins, and endocytosis is clathrin dependent. The vast majority of internalized GPI-anchored protein is efficiently recycled, while the processes by which transmembrane domain (TMD) proteins are internalized and sorted are unknown. We demonstrate that internalization of invariant surface glycoprotein (ISG)65, a trypanosome TMD protein, involves ubiquitylation and also requires clathrin. We find a hierarchical requirement for cytoplasmic lysine residues in internalization and turnover, and a single position-specific lysine is sufficient for degradation, surface removal and attachment of oligoubiquitin chains. Ubiquitylation is context dependent as provision of additional lysine residues by C-terminal fusion of neuronal precursor cell-expressed developmentally downregulated protein (NEDD)8 fails to support ubiquitylation. Attachment of NEDD8 leads to degradation by a second ubiquitin-independent pathway. Moreover, degradation of ubiquitylated or NEDDylated substrate takes place in an acidic compartment and is proteosome independent. Significantly, in non-opisthokont lineages, Rsp5p or c-Cbl, the E3 ubiquitin ligases acting on endocytic cargo, are absent but Uba1 class genes are present and are required for cell viability and ISG65 ubiquitylation. Hence, ubiquitylation is an evolutionarily conserved mechanism for internalization of surface proteins, but aspects of the machinery differ substantially between the major eukaryotic lineages.     

The Escherichia coli adherence factor plasmid of enteropathogenic Escherichia coli causes a global decrease in ubiquitylated host cell proteins by decreasing ubiquitin E1 enzyme expression through host aspartyl proteases

Ubiquitylation is a widespread post-translational global regulatory system that is essential for the proper functioning of various cellular events. Recent studies have shown that certain types of Escherichia coli can exploit specific aspects of the ubiquitylation system to influence downstream targets. Despite these findings, examination of the effects pathogenic E. coli have on the overall host ubiquitylation system remain unexplored. To study the impact that pathogenic E. coli have on the ubiquitylation levels of host proteins during infections, we analyzed the entire ubiquitylation system during enteropathogenic E. coli infections of cultured cells. We found that these microbes caused a dramatic decrease in ubiquitylated host proteins during these infections. This occurred with a concomitant reduction in the expression of essential E1 activating enzymes in the host, which are integral for the initiation of the ubiquitylation cascade. Control of host E1 enzyme levels was dependent on the E. coli adherence factor plasmid which acted on host aspartyl proteases within enteropathogenic E. coli. Hijacking of the ubiquitylation system did not require the plasmid-encoded regulator or bundle forming pilus expression, as enteropathogenic E. coli mutated in those factors did not revert the ubiquitylation of host proteins or the abundance of E1 enzyme proteins to uninfected levels. Our work shows that E. coli have developed strategies to usurp post-translational systems by targeting crucial enzymes. The ability of enteropathogenic E. coli to inactivate host protein ubiquitylation could enable more efficient effector protein functionality, providing increased bacterial control of host cells during enteropathogenic E. coli pathogenesis.     

Emerging roles for ubiquitin in adenovirus cell entry

Adenovirus relies on numerous interactions between viral and host cell proteins to efficiently enter cells. Undoubtedly, post-translational modifications of host and cellular proteins can impact the efficiency of this cell entry process. Ubiquitylation, once simply thought of as a modification targeting proteins for proteasomal degradation, is now known to regulate protein trafficking within cells, protein-protein interactions and cell signalling pathways. Accumulating evidence suggests that protein ubiquitylation can influence all stages of the life cycle of other viruses such as cell entry, replication and egress. Until recently, the influence of ubiquitylation has only been documented during adenovirus replication. This review highlights the most recent evidence demonstrating direct engagement of host ubiquitylation and SUMOylation machinery by adenovirus during cell entry. Additionally, potential roles for host protein ubiquitylation and the potential for adenovirus regulation of host ubiquitylation machinery during cell entry are discussed.     

Intersectin 1 enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2-Cbl interaction

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.     

The ubiquitin ligase Rsp5p is required for modification and sorting of membrane proteins into multivesicular bodies

Precursor forms of vacuolar proteins with transmembrane domains, such as the carboxypeptidase S Cps1p and the polyphosphatase Phm5p, are selectively sorted in endosomal compartments to vesicles that invaginate, budding into the lumen of the late endosomes, resulting in the formation of multivesicular bodies (MVBs). These proteins are then delivered to the vacuolar lumen following fusion of the MVBs with the vacuole. The sorting of Cps1p and Phm5p to these structures is mediated by ubiquitylation, and in doa4 mutant cells, which have reduced level of free ubiquitin, these proteins are missorted to the vacuolar membrane. A RING-finger ubiquitin ligase Tul1p has been shown to participate in the ubiquitylation of Cps1p and Phm5p. We show here that the HECT-ubiquitin ligase Rsp5p is also required for the ubiquitylation of these proteins, and therefore for their sorting to MVBs. Rsp5p is an essential ubiquitin ligase containing an N-terminal C2 domain followed by three WW domains, and a C-terminal catalytic HECT domain. In cells with low levels of Rsp5p (npi1 mutant cells), vacuolar hydrolases do not reach the vacuolar lumen and are instead missorted to the vacuolar membrane. The C2 domain and both the second and third WW domains of Rsp5p are important determinants for sorting to MVBs. Ubiquitylation of Cps1p was strongly reduced in the npi1 mutant strain and ubiquitylation was completely abolished in the npi1 tul1Delta double mutant. These data demonstrate that Rsp5p plays a novel and key role in intracellular trafficking, and extend the currently very short list of substrates ubiquitylated in vivo by several different ubiquitin ligases acting cooperatively.     

Ubiquitylation‐dependent oligomerization regulates activity of Nedd4 ligases

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac I_KS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1‐helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1‐conjugated ubiquitin and the HECT ubiquitin‐binding patch pulls the α1‐helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the I_KS channel, thus confirming the functional importance of E3‐ligase autoinhibition.     

Structural basis of ubiquitylation

The attachment of the small protein ubiquitin to other proteins, a process known as ubiquitylation, is a widespread form of post-translational modification that regulates numerous cellular functions in eukaryotes. Ubiquitylation is performed by complexes of E2 and E3 enzymes that are assembled and select substrates via a series of protein-protein interactions. Recent structure determinations of the ubiquitylation machinery have revealed some of the various protein-protein interfaces involved.     

A Highlights from MBoC Selection: A phosphoinositide-binding cluster in cavin1 acts as a molecular sensor for cavin1 degradation

Caveolae are abundant surface organelles implicated in a range of cellular processes. Two classes of proteins work together to generate caveolae: integral membrane proteins termed caveolins and cytoplasmic coat proteins called cavins. Caveolae respond to membrane stress by releasing cavins into the cytosol. A crucial aspect of this model is tight regulation of cytosolic pools of cavin under resting conditions. We now show that a recently identified region of cavin1 that can bind phosphoinositide (PI) lipids is also a major site of ubiquitylation. Ubiquitylation of lysines within this site leads to rapid proteasomal degradation. In cells that lack caveolins and caveolae, cavin1 is cytosolic and rapidly degraded as compared with cells in which cavin1 is associated with caveolae. Membrane stretching causes caveolar disassembly, release of cavin complexes into the cytosol, and increased proteasomal degradation of wild-type cavin1 but not mutant cavin1 lacking the major ubiquitylation site. Release of cavin1 from caveolae thus leads to exposure of key lysine residues in the PI-binding region, acting as a trigger for cavin1 ubiquitylation and down-regulation. This mutually exclusive PI-binding/ubiquitylation mechanism may help maintain low levels of cytosolic cavin1 in resting cells, a prerequisite for cavins acting as signaling modules following release from caveolae.     

Receptor Tyrosine Kinase Ubiquitylation Involves the Dynamic Regulation of Cbl-Spry2 by Intersectin 1 and the Shp2 Tyrosine Phosphatase

Ubiquitylation of receptor tyrosine kinases (RTKs) regulates their trafficking and lysosomal degradation. The multidomain scaffolding protein intersectin 1 (ITSN1) is an important regulator of this process. ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity is unclear. Here, we demonstrate that ITSN1 interacts with and recruits the Shp2 tyrosine phosphatase to Spry2 to enhance its dephosphorylation, thereby disrupting the inhibitory effect of Spry2 on Cbl and enhancing EGFR ubiquitylation. In contrast, expression of a catalytically inactive Shp2 mutant reversed the effect of ITSN1 on Spry2 dephosphorylation and decreased Cbl-mediated EGFR ubiquitylation. In addition, disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 resulted in decreased Shp2-Spry2 interaction and enhanced Spry2 tyrosine phosphorylation. This study demonstrates that ITSN1 enhances Cbl activity, in part, by modulating the interaction of Cbl with Spry2 through recruitment of Shp2 phosphatase to the Cbl-Spry2 complex. These findings reveal a new level of complexity in the regulation of RTKs by Cbl through ITSN1 binding with Shp2 and Spry2.