Michael Wallisch

A novel ubiquitin ligase is deficient in Fanconi anemia

Fanconi anemia is a recessively inherited disease characterized by congenital defects, bone marrow failure and cancer susceptibility. Cells from individuals with Fanconi anemia are highly sensitive to DNA-crosslinking drugs, such as mitomycin C (MMC). Fanconi anemia proteins function in a DNA damage response pathway involving breast cancer susceptibility gene products, BRCA1 and BRCA2 (refs. 1,2). A key step in this pathway is monoubiquitination of FANCD2, resulting in the redistribution of FANCD2 to nuclear foci containing BRCA1 (ref. 3). The underlying mechanism is unclear because the five Fanconi anemia proteins known to be required for this ubiquitination have no recognizable ubiquitin ligase motifs. Here we report a new component of a Fanconi anemia protein complex, called PHF9, which possesses E3 ubiquitin ligase activity in vitro and is essential for FANCD2 monoubiquitination in vivo. Because PHF9 is defective in a cell line derived from an individual with Fanconi anemia, we conclude that PHF9 (also called FANCL) represents a novel Fanconi anemia complementation group (FA-L). Our data suggest that PHF9 has a crucial role in the Fanconi anemia pathway as the likely catalytic subunit required for monoubiquitination of FANCD2.

A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome.

ABSTRACT Bloom syndrome (BS) is a genetic disorder associated with dwarfism, immunodeficiency, reduced fertility, and an elevated risk of cancer. To investigate the mechanism of this disease, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA proteins). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity, and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIα and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from an FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair.

Fanconi Anemia Proteins Are Required To Prevent Accumulation of Replication-Associated DNA Double-Strand Breaks

ABSTRACT Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

FANCD2 protein is expressed in proliferating cells of human tissues that are cancer-prone in Fanconi anaemia

Fanconi anaemia (FA) is an inherited form of progressive pancytopenia associated with developmental defects, chromosomal instability, and cancer predisposition. At least seven distinct FA proteins function in concert to protect the genome, a key step being the activation of FANCD2 by mono‐ubiquitination. This paper reports an immunohistochemical analysis of FANCD2 expression in normal human tissue. The highest expression was observed in maturing spermatocytes and fetal oocytes (consistent with a role for FANCD2 in meiosis) and in germinal centre cells of the spleen, tonsil, and lymph nodes (consistent with a role in proliferation). FANCD2 expression was also seen in tissues predisposed to cancer development in FA patients: haematopoietic cells, especially in the fetus, and squamous cell epithelia, particularly in the head and neck region and uterine cervix. FANCD2 expression was also occasionally seen in the breast and Fallopian tube epithelium, the respiratory epithelium of the trachea, and the exocrine cells of the pancreas, indicating that these tissues may also be cancer‐prone in FA. FANCD2 expression is frequently expressed in proliferating cells as demonstrated by Ki‐67 immunofluorescence double staining, consistent with a function of FANCD2 in DNA replication. Copyright

Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function

The Tec family kinase Brutons tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding.

Respiratory effects of chronic in utero methadone or morphine exposure in the neonatal guinea pig

This study uses a neonatal guinea pig model to compare the effects of in utero methadone or morphine exposure upon breathing control. We hypothesize that in utero methadone exposure will result in similar respiratory disturbances to those seen in morphine exposed neonates, but that the onset will be slower and the duration longer, due to methadones longer elimination half-life. Pregnant Dunkin-Hartley guinea pigs received once-daily injections of methadone, morphine, or vehicle (saline) during the last half of gestation and pups were studied 3, 7, or 14 days after birth. In utero methadone or morphine exposure resulted in decreased birth weight compared to vehicle, and pups experienced a withdrawal syndrome which included increased locomotor activity and respiratory disturbances but no change in rectal temperature. Both opioid exposures increased inspiratory minute ventilation during CO(2) challenge at 3 days after birth, but only in morphine exposed pups was this withdrawal effect still present on day 7. Surprisingly, only morphine exposure increased inspiratory minute ventilation during room air breathing. We conclude that in utero methadone exposure is not equivalent to in utero morphine exposure. With respect to neonatal respiratory control, methadone-induced changes in respiration are only apparent during hypercapnia.

A novel, non-invasive method of respiratory monitoring for use with stereotactic procedures

Accurate monitoring of respiration is often needed for neurophysiological studies, as either a dependent experimental variable or an indicator of physiological state. Current options for respiratory monitoring of animals held in a stereotaxic frame include EMG recordings, pneumotachograph measurements, inductance-plethysmography, whole-body plethysmography (WBP), and visual monitoring. While powerful, many of these methods prevent access to the animals body, interfere with experimental manipulations, or require deep anesthesia and additional surgery. For experiments where these issues may be problematic, we developed a non-invasive method of recording respiratory parameters specifically for use with animals held in a stereotaxic frame. This system, ventilation pressure transduction (VPT), measures variations in pressure at the animals nostril from inward and outward airflow during breathing. These pressure changes are detected by a sensitive pressure transducer, then filtered and amplified. The output is an analog signal representing each breath. VPT was validated against WBP using 10% carbon dioxide and systemic morphine (4mg/kg) challenges in lightly anesthetized animals. VPT accurately represented breathing rate and tidal volume changes under both baseline and challenge conditions. This novel technique can therefore be used to measure respiratory rate and relative tidal volume when stereotaxic procedures are needed for neuronal manipulations and recording.

Chronic in utero buprenorphine exposure causes prolonged respiratory effects in the guinea pig neonate

Our laboratory studies the effects of in utero opioid exposure on the neonate. In this work we test the effects of chronic in utero exposure to buprenorphine on the neonate. Buprenorphine is a promising candidate for treatment of opioid addiction during pregnancy and it has been suggested to decrease the neonatal abstinence syndrome in human infants. In our guinea pig model, we focused not only on the respiratory effects of in utero exposure on the neonate, but also studied withdrawal signs in the neonate, a major concern of all opioid treatment during pregnancy. Pregnant guinea pigs were treated with daily subcutaneous injections of 0.1mg/kg buprenorphine during the second half of gestation. We measured weight, locomotor activity and respiratory function in pups of ages 3 to 14 days. Respiratory response was recorded using a two-chamber plethysmograph, while pups were breathing either room air or 5% CO(2). Our results show that chronic in utero exposure to buprenorphine induces respiratory effects up to day 14 after birth, while earlier studies have shown that effects of either in utero methadone or morphine only persist in the first week after birth in the guinea pig model. These data provide important information for clinical trials of buprenorphine treatment suggesting that duration and severity of respiratory effects of in utero buprenorphine exposure should be monitored.

S-Methadone augments R-methadone induced respiratory depression in the neonatal guinea pig.

Methadone is administered as a racemic mixture, although its analgesic and respiratory effects are attributed to R-isomer activity at the mu opioid receptor (MOP). Recently, we observed a four-fold increase in inspiratory time in 3-day-old guinea pigs following an injection of racemic methadone. We hypothesized that this effect was due to augmentation of R-methadone induced respiratory depression by the S-methadone isomer. In the current longitudinal study, we injected 3-, 7-, and 14-day-old neonatal guinea pigs with saline, R-methadone, S-methadone, or R- plus S-methadone in order to characterize the roles of the individual isomers, as well as the synergistic effects of co-administration. Using plethysmography, we measured respiratory parameters while breathing room air and during a 5% CO(2) challenge. S-Methadone alone had no respiratory effects. However, the R- plus S-methadone group showed greater respiratory depression and increased inspiratory time than the R-methadone group in the youngest animals, suggesting that the respiratory effects of R-methadone are augmented by S-methadone in early development.

WEDGE: an anticoagulant thrombin mutant produced by autoactivation.

The production of therapeutically relevant proteases typically involves activation of a zymogen precursor by external enzymes, which may raise regulatory issues about availability and purity. Recent studies of thrombin precursors have shown how to engineer constructs that spontaneously convert to the mature protease by autoactivation, without the need for external enzymes.