The regulation of necroptosis by post-translational modifications

Necroptosis is a caspase-independent, lytic form of programmed cell death whose errant activation has been widely implicated in many pathologies. The pathway relies on the assembly of the apical protein kinases, RIPK1 and RIPK3, into a high molecular weight cytoplasmic complex, termed the necrosome, downstream of death receptor or pathogen detector ligation. The necrosome serves as a platform for RIPK3-mediated phosphorylation of the terminal effector, the MLKL pseudokinase, which induces its oligomerization, translocation to, and perturbation of, the plasma membrane to cause cell death. Over the past 10 years, knowledge of the post-translational modifications that govern RIPK1, RIPK3 and MLKL conformation, activity, interactions, stability and localization has rapidly expanded. Here, we review current knowledge of the functions of phosphorylation, ubiquitylation, GlcNAcylation, proteolytic cleavage, and disulfide bonding in regulating necroptotic signaling. Post-translational modifications serve a broad array of functions in modulating RIPK1 engagement in, or exclusion from, cell death signaling, whereas the bulk of identified RIPK3 and MLKL modifications promote their necroptotic functions. An enhanced understanding of the modifying enzymes that tune RIPK1, RIPK3, and MLKL necroptotic functions will prove valuable in efforts to therapeutically modulate necroptosis.Subject terms: Kinases, Proteomics, Cell biology     

Innate immunity: regulation of caspases by IAP-dependent ubiquitylation

Caspases are widely known as initiators and executioners of cell death. Full activation of caspases leading to cleavage of many cellular substrates was long considered to be a point-of-no-return in the apoptosis pathway. However, it also has been known that activated caspases do not always have the ability to kill, but instead initiate non-apoptotic processes such as cell differentiation or activation of innate immune responses. In this issue of The EMBO Journal, Meinander et al (2012) explore the contribution of polyubiquitination of Dredd, a known initiator caspase, to the activation of innate immunity. The authors show that infection with gram-negative bacteria leads to DIAP2-dependent ubiquitylation of Dredd which in turn is required for processing of Relish (Rel) and expression of antimicrobial peptide (AMP) genes that are indispensable for fighting the infection.     

Dynamic regulation of PCNA ubiquitylation/deubiquitylation

Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation plays a crucial role in maintaining genomic stability during DNA replication. DNA damage stalling the DNA replication fork induces PCNA ubiquitylation that activates DNA damage bypass to prevent the collapse of DNA replication forks that could potentially produce double-strand breaks and chromosomal rearrangements. PCNA ubiquitylation dictates the mode of bypass depending on the level of ubiquitylation; monoubiquitylation and polyubiquitylation activate error-prone translesion synthesis and error-free template switching, respectively. Due to the error-prone nature of DNA damage bypass, PCNA ubiquitylation needs to be tightly regulated. Here, we review the molecular mechanisms to remove ubiquitin from PCNA including the emerging role of USP1 and ELG1 in this fascinating process.     

程序性坏死(Necroptosis)的分子机制

程序性坏死(Necroptosis)是一种不同于凋亡及传统坏死的细胞程序性死亡方式, 可由肿瘤坏死因子受体(Tumor necrosis factor receptor, TNFR)或模式识别受体(Pattern recognition receptor, PRR)调控启动。受体相互作用蛋白(Receptor-interacting protein, RIP)1和3是启动necroptosis的两个关键蛋白, necroptosis启动后需要一系列分子传递和执行死亡信号, 如多核苷酸二磷酸-核糖聚合酶-1(Poly(ADP-ribose) polymerase, PARP-1)、活性氧簇(Reactive oxygen species, ROS)、Ca²⁺等, 这些分子破坏线粒体及其他细胞器, 最终使细胞在缺乏天冬氨酸半胱氨酸蛋白酶(Caspase)的情况下死亡。Necroptosis细胞可将损伤相关模式分子(Damage-associated molecular patterns, DAMPs)暴露到细胞外, 被吞噬细胞识别并清除。文章对启动necroptosis的受体分子、传递执行细胞坏死的重要分子和坏死细胞的清除过程进行了概述。     

Regulation of DNA repair by ubiquitylation

Cellular DNA repair is a frontline system that is responsible for maintaining genome integrity and thus preventing premature aging and cancer by repairing DNA lesions and strand breaks caused by endogenous and exogenous mutagens. However, it is also the principal cellular system in cancer cells that counteracts the killing effect of the major cancer treatments, e.g. chemotherapy and ionizing radiation. Although it is clear that an individual’s DNA repair capacity varies, the mechanisms involved in the regulation of repair systems that are responsible for such variations are only just emerging. This knowledge gap is impeding the finding of new cancer therapy targets and the development of novel treatment strategies. In recent years the vital role of post-translational modifications of DNA repair proteins, including ubiquitylation and phosphorylation, has been uncovered. This review will cover recent progress in our understanding of the role of ubiquitylation in the regulation of DNA repair.     

Regulation of DNA repair by ubiquitylation

The process of ubiquitylation is best known for its role in targeting proteins for degradation by the proteasome. However, recent studies of DNA-repair and DNA-damage-response pathways have significantly broadened the scope of the role of ubiquitylation to include non-proteolytic functions of ubiquitin. These pathways involve the monoubiquitylation of key DNA-repair proteins that have regulatory functions in homologous recombination and translesion DNA synthesis, and involve the polyubiquitylation of nucleotide-excision-repair proteins.     

c-Cbl mediated ubiquitylation and regulation of cell surface exposure of CD5

Downregulation of cell surface receptors is an important process aimed at attenuation or termination of receptor signaling. c-Cbl role in the process is thought to be initial ubiquitylation of the receptors targeted for degradation and assembly of internalization complexes consisting of several other proteins. c-Cbl seems to be present during the whole process of vesicle sorting after internalization. However, there are very few receptor molecules so far like EGFR being proven to be regulated by c-Cbl.It is known that a level of CD5 on mouse c-Cbl−/− thymocytes is upregulated in comparison to wild type cells. The mechanism leading to the upregulation is unknown. We show that CD5 is ubiquitylated in Jurkat-TAg cells and in mouse thymocytes and that the ubiquitylation is c-Cbl dependent. We also show that amount of CD5 associated with lysosomal marker LAMP-1 after stimulation is significantly lower in c-Cbl −/− thymocytes. CD5 mRNA level did not differ significantly between c-Cbl −/− and wild type thymocytes. We conclude that CD5 is ubiquitylated; the ubiquitylation is mediated by c-Cbl; CD5 level on a T lymphocyte cell surface is regulated by ubiquitylation and targeting to lysosomes.     

Improving bioorthogonal protein ubiquitylation by click reaction

Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase β, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.     

Regulation of base excision repair proteins by ubiquitylation

Human cellular DNA is under constant attack from both endogenous and exogenous mutagens, and consequently the base excision repair (BER) pathway plays a vital role in repairing damaged DNA bases, sites of base loss (apurinic/apyrimidinic sites) and DNA single strand breaks of varying complexity. BER thus maintains genome stability, and prevents the development of human diseases, such as premature aging, neurodegenerative diseases and cancer. Indeed, there is accumulating evidence that misregulation of BER protein levels is observed in cells and tissues from patients with these diseases, and that post-translational modifications, particularly ubiquitylation, perform a key role in controlling BER protein stability. This review will summarise the presently available data on ubiquitylation of some of the key BER proteins, and the functional consequences of this modification.     

The role of ubiquitylation in nerve cell development

Nerve cell development in the brain is a tightly regulated process. The generation of neurons from precursor cells, their migration to the appropriate target sites, their extensive arborization and their integration into functional networks through synapse formation and refinement are governed by multiple interdependent signalling cascades. The function and turnover of proteins involved in these signalling cascades, in turn, are spatially and temporally controlled by ubiquitylation. Recent advances have provided first insights into the highly complex and intricate molecular pathways that regulate ubiquitylation during all stages of neural development and that operate in parallel with other regulatory processes such as phosphorylation or cyclic nucleotide signalling.     

Necroptosis信号通路与靶向治疗的研究进展

Necroptosis是一种可调控的细胞程序性坏死途径,它具有与不可调控性细胞坏死相同的形态学特征。Necroptosis是caspase非依赖的。当细胞凋亡被阻断时,necroptosis信号通路由死亡结构域激活启动,其中RIP1的活化是necroptosis的关键步骤,该步骤可被necrostatin-1特异性抑制。近期研究表明,necroptosis在缺血性损伤、神经退行性疾病、恶性肿瘤、病毒感染和免疫性疾病等多种疾病的病理生理过程中起重要作用,有望作为药物开发的新靶点。对necroptosis的发现历程、信号通路及其在疾病病理生理机制中的作用和靶向necroptosis的治疗等四个方面进行综述。     

The regulation of necroptosis and perspectives for the development of new drugs preventing ischemic/reperfusion of cardiac injury

Apoptosis - In the last 10 years, mortality from acute myocardial infarction (AMI) has not significantly decreased. This situation is associated with the absence in clinical practice of...