The WD40-Repeat Protein-Containing Deubiquitinase Complex: Catalysis, Regulation, and Potential for Therapeutic Intervention

Ubiquitination has emerged as an essential signaling mechanism in eukaryotes. Deubiquitinases (DUBs) counteract the activities of the ubiquitination machinery and provide another level of control in cellular ubiquitination. Not surprisingly, DUBs are subjected to stringent regulations. Besides regulation by the noncatalytic domains present in the DUB sequences, DUB-interacting proteins are increasingly realized as essential regulators for DUB activity and function. This review focuses on DUBs that are associated with WD40-repeat proteins. Many human ubiquitin-specific proteases (USPs) were found to interact with WD40-repeat proteins, but little is known as to how this interaction regulates the activity and function of USPs. In recent years, significant progress has been made in understanding a prototypical WD40-repeat protein-containing DUB complex that comprises USP1 and USP1-associated factor 1 (UAF1). It has been shown that UAF1 activates USP1 through a potential active-site modulation, and the complex formation between USP1 and UAF1 is regulated by serine phosphorylation. Recently, human USPs have been recognized as a promising target class for inhibitor discovery. Small molecule inhibitors targeting several human USPs have been reported. USP1 is involved in two major DNA damage response pathways, DNA translesion synthesis and the Fanconi anemia pathway. Inhibiting the USP1/UAF1 deubiquitinase complex represents a new strategy to potentiate cancer cells to DNA-crosslinking agents and to overcome resistance that has plagued clinical cancer chemotherapy. The progress in inhibitor discovery against USPs and the WD40-repeat protein-containing USP complex will be discussed.     

ATM-mediated stabilization of hMutL DNA mismatch repair proteins augments p53 activation during DNA damage

Human DNA mismatch repair (MMR) proteins correct DNA errors and regulate cellular response to DNA damage by signaling apoptosis. Mutations of MMR genes result in genomic instability and cancer development. Nonetheless, how MMR proteins are regulated has not yet been determined. While hMLH1, hPMS2, and hMLH3 are known to participate in MMR, the function of another member of MutL-related proteins, hPMS1, remains unclear. Here we show that DNA damage induces the accumulation of hPMS1, hPMS2, and hMLH1 through ataxia-telangiectasia-mutated (ATM)-mediated protein stabilization. The subcellular localization of PMS proteins is also regulated during DNA damage, which induces nuclear localization of hPMS1 and hPMS2 in an hMLH1-dependent manner. The induced levels of hMLH1 and hPMS1 are important for the augmentation of p53 phosphorylation by ATM in response to DNA damage. These observations identify hMutL proteins as regulators of p53 response and demonstrate for the first time a function of hMLH1-hPMS1 complex in controlling the DNA damage response.     

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.     

Regulation of the Cell Division Cycle in Trypanosoma brucei

The cell division cycle is tightly regulated by the activation and inactivation of a series of proteins that control the replication and segregation of organelles to the daughter cells. During the past decade, we have witnessed significant advances in our understanding of the cell cycle in Trypanosoma brucei and how the cycle is regulated by various regulatory proteins. However, many other regulators, especially those unique to trypanosomes, remain to be identified, and we are just beginning to delineate the signaling pathways that drive the transitions through different cell cycle stages, such as the G₁/S transition, G₂/M transition, and mitosis-cytokinesis transition. Trypanosomes appear to employ both evolutionarily conserved and trypanosome-specific molecules to regulate the various stages of its cell cycle, including DNA replication initiation, spindle assembly, chromosome segregation, and cytokinesis initiation and completion. Strikingly, trypanosomes lack some crucial regulators that are well conserved across evolution, such as Cdc6 and Cdt1, which are involved in DNA replication licensing, the spindle motor kinesin-5, which is required for spindle assembly, the central spindlin complex, which has been implicated in cytokinesis initiation, and the actomyosin contractile ring, which is located at the cleavage furrow. Conversely, trypanosomes possess certain regulators, such as cyclins, cyclin-dependent kinases, and mitotic centromere-associated kinesins, that are greatly expanded and likely play diverse cellular functions. Overall, trypanosomes apparently have integrated unique regulators into the evolutionarily conserved pathways to compensate for the absence of those conserved molecules and, additionally, have evolved certain cell cycle regulatory pathways that are either different from its human host or distinct between its own life cycle forms.     

New friends for Ago2 in neuronal plasticity

MicroRNAs have emerged as central regulators of cellular homeostasis and increasing evidence suggests that they play a key role in neuronal plasticity. Major efforts are made to define microRNA networks and their targets in the brain. The mechanisms by which microRNA activity is regulated are, however, relatively unexplored. In this issue of The EMBO Journal, Störchel et al ( 2015 ) screened for proteins that affect microRNA function in neurons. They identify Nova1 and Ncoa3 as novel regulators of miRNA activity and demonstrate that both proteins are essential for neuronal plasticity in a microRNA‐dependent manner.