Human DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is crucial to the maintenance of genome integrity. The extreme infidelity of Polι and the fact that both its up- and down-regulation correlate with various cancers indicate that Polι expression and access to the replication fork should be strictly controlled. Here, we identify RNF2, an E3 ubiquitin ligase, as a new interacting partner of Polι that is responsible for Polι stabilization in vivo. Interestingly, while we report that RNF2 does not directly ubiquitinate Polι, inhibition of the E3 ubiquitin ligase activity of RNF2 affects the cellular level of Polι thereby protecting it from destabilization. Additionally, we indicate that this mechanism is more general, as DNA polymerase η, another Y-family polymerase and the closest paralogue of Polι, share similar features.

Protein ubiquitylation is an essential post-translational modification that regulates nearly all aspects of eukaryotic cell biology. A diverse collection of ubiquitylation signals, including an extensive repertoire of polymeric ubiquitin chains, leads to a range of different functional outcomes for the target protein. Recent studies have shown that ubiquitin chains can be branched and that branched chains have a direct impact on the stability or the activity of the target proteins they are attached to. In this mini review, we discuss the mechanisms that control the assembly and disassembly of branched chains by the enzymes of the ubiquitylation and deubiquitylation machinery. Existing knowledge regarding the activities of chain branching ubiquitin ligases and the deubiquitylases responsible for cleaving branched chains is summarized. We also highlight new findings concerning the formation of branched chains in response to small molecules that induce the degradation of otherwise stable proteins and examine the selective debranching of heterotypic chains by the proteasome-bound deubiquitylase UCH37.

During prolonged mitotic arrest induced by antimicrotubule drugs, cell fate decision is determined by two alternative pathways, one leading to cell death and the other inducing premature escape from mitosis by mitotic slippage. FBWX7, a member of the F-box family of proteins and substrate-targeting subunit of the SKP1-CUL1-F-Box E3 ubiquitin ligase complex, promotes mitotic cell death and prevents mitotic slippage, but molecular details underlying these roles for FBWX7 are unclear. In this study, we report that WDR5 (WD-repeat containing protein 5), a component of the mixed lineage leukemia complex of histone 3 lysine 4 methyltransferases, is a substrate of FBXW7. We determined by coimmunoprecipitation experiments and in vitro binding assays that WDR5 interacts with FBXW7 in vivo and in vitro. SKP1-CUL1-F-Box-FBXW7 mediates ubiquitination of WDR5 and targets it for proteasomal degradation. Furthermore, we find that WDR5 depletion counteracts FBXW7 loss of function by reducing mitotic slippage and polyploidization. In conclusion, our data elucidate a new mechanism in mitotic cell fate regulation, which might contribute to prevent chemotherapy resistance in patients after antimicrotubule drug treatment.

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Post-translational modifications, such as ubiquitylation, need to be tightly controlled to guarantee the accurate localization and activity of proteins. Ubiquitylation is a dynamic process primarily responsible for proteasome-mediated degradation of substrate proteins and crucial for both normal homeostasis and disease. Alterations in ubiquitylation lead to the upregulation of oncoproteins and/or downregulation of tumor suppressors, thus concurring in tumorigenesis. PROteolysis-TArgeting Chimera (PROTAC) is an innovative strategy that takes advantage by the cell\'s own Ubiquitin-Proteasome System (UPS). Each PROTAC molecule is composed by a ligand that recruits the target protein of interest (POI), a ligand specific for an E3 ubiquitin ligase enzyme, and a linker that connects these units. Upon binding to the POI, the PROTAC recruits the E3 inducing ubiquitylation-dependent proteasome degradation of the POI. To date, PROTAC technology has entered in clinical trials for several human cancers. Here, we will discuss the advantages and limitations of PROTACs development and safety considerations for their clinical application. Furthermore, we will review the potential of PROTAC strategy as therapeutic option in brain tumor, focusing on glioblastoma.

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Legionella pneumophila is a facultative intracellular pathogen that uses the Dot/Icm Type IV secretion system (T4SS) to translocate many effectors into its host and establish a safe, replicative lifestyle. The bacteria, once phagocytosed, reside in a vacuolar structure known as the Legionella-containing vacuole (LCV) within the host cells and rapidly subvert organelle trafficking events, block inflammatory responses, hijack the host ubiquitination system, and abolish apoptotic signaling. This arsenal of translocated effectors can manipulate the host factors in a multitude of different ways. These proteins also contribute to bacterial virulence by positively or negatively regulating the activity of one another. Such effector-effector interactions, direct and indirect, provide the delicate balance required to maintain cellular homeostasis while establishing itself within the host. This review summarizes the recent progress in our knowledge of the structure-function relationship and biochemical mechanisms of select effector pairs from Legionella that work in opposition to one another, while highlighting the diversity of biochemical means adopted by this intracellular pathogen to establish a replicative niche within host cells.

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound \"OFF\" state and GTP-bound \"ON\" state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound \"ON\" state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24-48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP.