Sarah L. Maslen

Regulatory Control of the Resolution of DNA Recombination Intermediates during Meiosis and Mitosis

The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activation. Mus81-Mms4 is hyperactivated by Cdc5-mediated phosphorylation in meiosis I, generating the crossovers necessary for chromosome segregation. Yen1 is also tightly regulated and is activated in meiosis II to resolve persistent Holliday junctions. In yeast and human mitotic cells, a similar regulatory network restrains these nuclease activities until mitosis, biasing the outcome of recombination toward noncrossover products while also ensuring the elimination of any persistent joint molecules. Mitotic regulation thereby facilitates chromosome segregation while limiting the potential for loss of heterozygosity and sister-chromatid exchanges.

FANCM and FAAP24 Function in ATR-Mediated Checkpoint Signaling Independently of the Fanconi Anemia Core Complex

The Fanconi anemia (FA) pathway is implicated in DNA repair and cancer predisposition. Central to this pathway is the FA core complex, which is targeted to chromatin by FANCM and FAAP24 following replication stress. Here we show that FANCM and FAAP24 interact with the checkpoint protein HCLK2 independently of the FA core complex. In addition to defects in FA pathway activation, downregulation of FANCM or FAAP24 also compromises ATR/Chk1-mediated checkpoint signaling, leading to defective Chk1, p53, and FANCE phosphorylation; 53BP1 focus formation; and Cdc25A degradation. As a result, FANCM and FAAP24 deficiency results in increased endogenous DNA damage and a failure to efficiently invoke cell-cycle checkpoint responses. Moreover, we find that the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, is required for its role in ATR/Chk1 signaling. Our data suggest that DNA damage recognition and remodeling activities of FANCM and FAAP24 cooperate with ATR/Chk1 to promote efficient activation of DNA damage checkpoints.

CK2 phospho-dependent binding of R2TP complex to TEL2 is essential for mTOR and SMG1 stability.

TEL2 interacts with and is essential for the stability of all phosphatidylinositol 3-kinase-related kinases (PIKKs), but its mechanism of action remains unclear. Here, we show that TEL2 is constitutively phosphorylated on conserved serines 487 and 491 by casein kinase 2 (CK2). Proteomic analyses establish that the CK2 phosphosite of TEL2 confers binding to the R2TP/prefoldin-like complex, which possesses chaperon/prefoldin activities required during protein complex assembly. The PIH1D1 subunit of the R2TP complex binds directly to the CK2 phosphosite of TEL2 in vitro and is required for the TEL2-R2TP/prefoldin-like complex interaction in vivo. Although the CK2 phosphosite mutant of TEL2 retains association with the PIKKs and HSP90 in cells, failure to interact with the R2TP/prefoldin-like complex results in instability of the PIKKs, principally mTOR and SMG1. We propose that TEL2 acts as a scaffold to coordinate the activities of R2TP/prefoldin-like and HSP90 chaperone complexes during the assembly of the PIKKs.

Plk1 and CK2 act in concert to regulate Rad51 during DNA double strand break repair.

Summary Homologous recombination (HR) plays an important role in the maintenance of genome integrity. HR repairs broken DNA during S and G2 phases of the cell cycle but its regulatory mechanisms remain elusive. Here, we report that Polo-like kinase 1 (Plk1), which is vital for cell proliferation and is frequently upregulated in cancer cells, phosphorylates the essential Rad51 recombinase at serine 14 (S14) during the cell cycle and in response to DNA damage. Strikingly, S14 phosphorylation licenses subsequent Rad51 phosphorylation at threonine 13 (T13) by casein kinase 2 (CK2), which in turn triggers direct binding to the Nijmegen breakage syndrome gene product, Nbs1. This mechanism facilitates Rad51 recruitment to damage sites, thus enhancing cellular resistance to genotoxic stresses. Our results uncover a role of Plk1 in linking DNA damage recognition with HR repair and suggest a molecular mechanism for cancer development associated with elevated activity of Plk1.

Dehydration-regulated processing of late embryogenesis abundant protein in a desiccation-tolerant nematode

Late embryogenesis abundant (LEA) proteins occur in desiccation‐tolerant organisms, including the nematode Aphelenchus avenae, and are thought to protect other proteins from aggregation. Surprisingly, expression of the LEA protein AavLEA1 in A. avenae is partially discordant with that of its gene: protein is present in hydrated animals despite low cognate mRNA levels. Moreover, on desiccation, when its gene is upregulated, AavLEA1 is specifically cleaved to discrete, smaller polypeptides. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes, and main cleavage sites were mapped to 11‐mer repeated motifs in the AavLEA1 sequence. Processed polypeptides retain function as protein anti‐aggregants and we hypothesise that the expression pattern and cleavage of LEA protein allow rapid, maximal availability of active molecules to the dehydrating animal.

Molecular basis of APC/C regulation by the spindle assembly checkpoint.

In the dividing eukaryotic cell, the spindle assembly checkpoint (SAC) ensures that each daughter cell inherits an identical set of chromosomes. The SAC coordinates the correct attachment of sister chromatid kinetochores to the mitotic spindle with activation of the anaphase-promoting complex (APC/C), the E3 ubiquitin ligase responsible for initiating chromosome separation. In response to unattached kinetochores, the SAC generates the mitotic checkpoint complex (MCC), which inhibits the APC/C and delays chromosome segregation. By cryo-electron microscopy, here we determine the near-atomic resolution structure of a human APC/C–MCC complex (APC/CMCC). Degron-like sequences of the MCC subunit BubR1 block degron recognition sites on Cdc20, the APC/C coactivator subunit responsible for substrate interactions. BubR1 also obstructs binding of the initiating E2 enzyme UbcH10 to repress APC/C ubiquitination activity. Conformational variability of the complex enables UbcH10 association, and structural analysis shows how the Cdc20 subunit intrinsic to the MCC (Cdc20MCC) is ubiquitinated, a process that results in APC/C reactivation when the SAC is silenced.

Molecular Mechanism of Apc/C Activation by Mitotic Phosphorylation.

In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4, 5, 6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk–cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/CCdc20 rather than APC/CCdh1, and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.

Peptide and small molecule inhibitors of HECT-type ubiquitin ligases

Significance The ubiquitin system controls a wide range of processes in cells and provides attractive drug targets for the treatment of cancer and other diseases. However, it has proved difficult to obtain inhibitors of the ligases that conjugate ubiquitin to substrates, of which there are hundreds. One class, the HECT (homologous to E6AP C terminus) domain ligases, receives ubiquitin from an E2 enzyme and transfers it to substrate. We have selected bicyclic peptides that block the E2 binding site of individual HECT ligases, as well as a small molecule, heclin (HECT ligase inhibitor), that broadly inhibits these ligases in cells. These inhibitors demonstrate that HECT domains are druggable targets and provide tools to study ubiquitination. The same approach could be used to select further HECT inhibitors. The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502–507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.

Clusterin secreted by astrocytes enhances neuronal differentiation from human neural precursor cells

Neuronal differentiation from expanded human ventral mesencephalic neural precursor cells (NPCs) is very limited. Astrocytes are known to secrete neurotrophic factors, and so in order to enhance neuronal survival from NPCs, we tested the effect of regional astrocyte-conditioned medium (ACM) from the rat cortex, hippocampus and midbrain on this process. Human NPCs were expanded in FGF-2 before differentiation for 1 or 4 weeks in ACM. The results show that ACM from the hippocampus and midbrain increase the number of neurons from expanded human NPCs, an effect that was not observed with cortical ACM. In addition, both hippocampal and midbrain ACM increased the number and length of phosphorylated neurofilaments. MALDI-TOF analysis used to determine differences in media revealed that although all three regional ACMs had cystatin C, α-2 macroglobulin, extracellular matrix glycoprotein and vimentin, only hippocampal and midbrain ACM also contained clusterin, which when immunodepleted from midbrain ACM eliminated the observed effects on neuronal differentiation. Furthermore, clusterin is a highly glycosylated protein that has no effect on cell proliferation but decreases apoptotic nuclei and causes a sustained increase in phosphorylated extracellular signal-regulated kinase, implicating its role in cell survival and differentiation. These findings further reveal differential effects of regional astrocytes on NPC behavior and identify clusterin as an important mediator of NPC-derived neuronal survival and differentiation.

The Synthesis of Azadirachtin: A Potent Insect Antifeedant

We describe in full the first synthesis of the potent insect antifeedant azadirachtin through a highly convergent approach. An O-alkylation reaction is used to unite decalin ketone and propargylic mesylate fragments, after which a Claisen rearrangement constructs the central C8-C14 bond in a stereoselective fashion. The allene which results from this sequence then enables a second critical carbon-carbon bond forming event whereby the [3.2.1] bicyclic system, present in the natural product, is generated via a 5-exo-radical cyclisation process. Finally, using knowledge gained through our early studies into the reactivity of the natural product, a series of carefully designed steps completes the synthesis of this challenging molecule.