A novel de novo truncating variant in a Hungarian patient with CTNNB1 neurodevelopmental disorder.

PURPOSE: We aimed to elucidate the underlying disease in a Hungarian family, with only one affected family member, a 16-year-old male Hungarian patient, who developed global developmental delay, cognitive impairment, behavioral problems, short stature, intermittent headaches, recurrent dizziness, strabismus, hypermetropia, complex movement disorder and partial pituitary dysfunction. After years of detailed clinical investigations and careful pediatric care, the exact diagnosis of the patient and the cause of the disease was still unknown. METHODS: We aimed to perform whole exome sequencing (WES) in order to investigate whether the affected patient is suffering from a rare monogenic disease. RESULTS: Using WES, we identified a novel, de novo frameshift variant (c.1902dupG, p.Ala636SerfsTer12) of the catenin beta-1 (CTNNB1) gene. Assessment of the novel CTNNB1 variant suggested that it is a likely pathogenic one and raised the diagnosis of CTNNB1 neurodevelopmental disorder (OMIM 615,075). CONCLUSIONS: Our manuscript may contribute to the better understanding of the genetic background of the recently discovered CTNNB1 neurodevelopmental disorder and raise awareness among clinicians and geneticists. The affected Hungarian family demonstrates that based on the results of the clinical workup is difficult to establish the diagnosis and high-throughput genetic screening may help to solve these complex cases.

Missing Heritability in Albinism: Deep Characterization of a Hungarian Albinism Cohort Raises the Possibility of the Digenic Genetic Background of the Disease.

Albinism is characterized by a variable degree of hypopigmentation affecting the skin and the hair, and causing ophthalmologic abnormalities. Its oculocutaneous, ocular and syndromic forms follow an autosomal or X-linked recessive mode of inheritance, and 22 disease-causing genes are implicated in their development. Our aim was to clarify the genetic background of a Hungarian albinism cohort. Using a 22-gene albinism panel, the genetic background of 11 of the 17 Hungarian patients was elucidated. In patients with unidentified genetic backgrounds (n = 6), whole exome sequencing was performed. Our investigations revealed a novel, previously unreported rare variant (N687S) of the two-pore channel two gene (TPCN2). The N687S variant of the encoded TPC2 protein is carried by a 15-year-old Hungarian male albinism patient and his clinically unaffected mother. Our segregational analysis and in vitro functional experiments suggest that the detected novel rare TPCN2 variant alone is not a disease-causing variant in albinism. Deep genetic analyses of the family revealed that the patient also carries a phenotype-modifying R305W variant of the OCA2 protein, and he is the only family member harboring this genotype. Our results raise the possibility that this digenic combination might contribute to the observed differences between the patient and the mother, and found the genetic background of the disease in his case.

Beyond C9orf72: repeat expansions and copy number variations as risk factors of amyotrophic lateral sclerosis across various populations.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder which is characterized by the loss of both upper and lower motor neurons in the central nervous system. In a significant fraction of ALS cases - irrespective of family history- a genetic background may be identified. The genetic background of ALS shows a high variability from one ethnicity to another. The most frequent genetic cause of ALS is the repeat expansion of the C9orf72 gene. With the emergence of next-generation sequencing techniques and copy number alteration calling tools the focus in ALS genetics has shifted from disease causing genes and mutations towards genetic susceptibility and risk factors.In this review we aimed to summarize the most widely recognized and studied ALS linked repeat expansions and copy number variations other than the hexanucleotide repeat expansion in the C9orf72 gene. We compare and contrast their involvement and phenotype modifying roles in ALS among different populations.

Whole-Exome Sequencing Identified Two Novel Pathogenic Mutations in the PTCH1 Gene in BCNS.

Basal cell nevus syndrome (BCNS, OMIM 109400) is a familial cancer syndrome characterized by the development of numerous basal cell cancers and various other developmental abnormalities, including epidermal cysts of the skin, calcified dural folds, keratocysts of the jaw, palmar and plantar pits, ovarian fibromas, medulloblastomas, lymphomesenteric cysts, and fetal rhabdomyomas. BCNS shows autosomal dominant inheritance and is caused by mutations in the patched 1 (PTCH1) gene and the suppressor of the fused homolog (SUFU) gene. In a few cases, variants of patched 2 (PTCH2) have been found in patients who met the criteria for BCNS. In an investigation of 11 Hungarian families who fulfilled the diagnostic criteria for BCNS, whole-exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA) identified two novel pathogenic variants (c.2994C>A; p.Cys998Ter and c.814_818del; p.Asn272SerfsTer11), one recently identified variant (c.1737_1745del p.Val580_Val582del), and three recurrent disease-causing variants of the PTCH1 gene with a diagnosis rate of 63.6%. Disease-causing variants were not found for the SUFU and PTCH2 genes. These applied methods could not fully elucidate the genetic background of all the BCNS cases that we investigated. To uncover the missing heritability of BCNS, whole-genome sequencing or an epigenetic approach might be considered in the future.

Genetic Screening of a Hungarian Cohort with Focal Dystonia Identified Several Novel Putative Pathogenic Gene Variants.

Dystonia is a rare movement disorder which is characterized by sustained or intermittent muscle contractions causing abnormal and often repetitive movements, postures, or both. The two most common forms of adult-onset focal dystonia are cervical dystonia (CD) and benign essential blepharospasm (BSP). A total of 121 patients (CD, 74; BSP, 47) were included in the study. The average age of the patients was 64 years. For the next-generation sequencing (NGS) approach, 30 genes were selected on the basis of a thorough search of the scientific literature. Assessment of 30 CD- and BSP-associated genes from 121 patients revealed a total of 209 different heterozygous variants in 24 genes. Established clinical and genetic validity was determined for nine heterozygous variations (three likely pathogenic and six variants of uncertain significance). Detailed genetic examination is an important part of the work-up for focal dystonia forms. To our knowledge, our investigation is the first such study to be carried out in the Middle-European region.

Genetic Etiology of Nonsyndromic Hearing Loss in Hungarian Patients.

Hearing loss is the most prevalent sensory disorder worldwide. The majority of congenital nonsyndromic hearing loss (NSHL) cases are caused by hereditary factors. Previously, the majority of NSHL studies focused on the GJB2 gene; however, with the availability of next-generation sequencing (NGS) methods, the number of novel variants associated with NSHL has increased. The purpose of this study was to design effective genetic screening for a Hungarian population based on a pilot study with 139 NSHL patients. A stepwise, comprehensive genetic approach was developed, including bidirectional capillary sequencing, multiplex ligation-dependent probe amplification (MLPA), and an NGS panel of 108 hearing loss genes. With our results, a genetic diagnosis was possible for 92 patients. Sanger sequencing and MLPA identified the genetic background of 50% of these diagnosed cases, and the NGS panel identified another 16%. The vast majority (92%) of the diagnosed cases showed autosomal recessive inheritance and 76% were attributed to GJB2. The implementation of this stepwise analysis markedly increased our diagnostic yield and proved to be cost-effective as well.

Likely Pathogenic Variants of Cav1.3 and Nav1.1 Encoding Genes in Amyotrophic Lateral Sclerosis Could Elucidate the Dysregulated Pain Pathways.

Amyotrophic lateral sclerosis (ALS) is a lethal multisystem neurodegenerative disease associated with progressive loss of motor neurons, leading to death. Not only is the clinical picture of ALS heterogenous, but also the pain sensation due to different types of pain involvement. ALS used to be considered a painless disease, but research has been emerging and depicting a more complex pain representation in ALS. Pain has been detected even a couple years before the symptomatic stage of ALS, referring to primary pain associated with muscle denervation, although secondary pain due to nociceptive causes is also a part of the clinical picture. A new non-contact dying-back injury mechanism theory of ALS recently postulated that the irreversible intrafusal proprioceptive Piezo2 microinjury could be the primary damage, with underlying genetic and environmental risk factors. Moreover, this Piezo2 primary damage is also proposed to dysregulate the primary pain pathways in the spinal dorsal horn in ALS due to the lost imbalanced subthreshold Ca2+ currents, NMDA activation and lost L-type Ca2+ currents, leading to the lost activation of wide dynamic range neurons. Our investigation is the first to show that the likely pathogenic variants of the Cav1.3 encoding CACNA1D gene may play a role in ALS pathology and the associated dysregulation or loss of the pain sensation. Furthermore, our reanalysis also shows that the SCN1A gene might also contribute to the dysregulated pain sensation in ALS. Finally, the absence of pathogenic variants of Piezo2 points toward the new non-contact dying-back injury mechanism theory of ALS. However, molecular and genetic investigations are needed to identify the functionally diverse features of this proposed novel critical pathway.

STABILON, a Novel Sequence Motif That Enhances the Expression and Accumulation of Intracellular and Secreted Proteins.

The dynamic balance of transcriptional and translational regulation together with degron-controlled proteolysis shapes the ever-changing cellular proteome. While a large variety of degradation signals has been characterized, our knowledge of cis-acting protein motifs that can in vivo stabilize otherwise short-lived proteins is very limited. We have identified and characterized a conserved 13-mer protein segment derived from the p54/Rpn10 ubiquitin receptor subunit of the Drosophila 26S proteasome, which fulfills all the characteristics of a protein stabilization motif (STABILON). Attachment of STABILON to various intracellular as well as medically relevant secreted model proteins resulted in a significant increase in their cellular or extracellular concentration in mammalian cells. We demonstrate that STABILON acts as a universal and dual function motif that, on the one hand, increases the concentration of the corresponding mRNAs and, on the other hand, prevents the degradation of short-lived fusion proteins. Therefore, STABILON may lead to a breakthrough in biomedical recombinant protein production.

Re-analysis of the Hungarian amyotrophic lateral sclerosis population and evaluation of novel ALS genetic risk variants.

Amyotrophic lateral sclerosis (ALS) is a presently incurable neurodegenerative disease. Some genes have a causal relationship to ALS, others act as susceptibility and/or risk factors. We aimed to elucidate the role of 14 ALS-related genes in the Hungarian ALS population of 183 patients. Mutation screening of major ALS genes was performed. SMN1 and SMN2 genes were examined by multiplex ligation-dependent probe-amplification assay; intermediate repeat expansions in the ATXN1 and ATXN2 genes were analyzed by fragment analysis. Additional variants in putative ALS genes were screened from previously acquired next generation sequencing data. We confirmed the repeat expansion of the C9orf72, ATXN1 and ATXN2 genes as ALS risk factors in this Hungarian cohort. Additionally, we identified a pathogenic SOD1 mutation and suggested its founder effect. A likely pathogenic variant in the MFSD8 gene was detected, and variants of interest were uncovered in the ANXA11 and GLT8D1 genes. We provide valuable data as part of the growing body of work on population-specific aspects of the genetic background of ALS.

Usp14 is required for spermatogenesis and ubiquitin stress responses in Drosophila melanogaster.

Deubiquitylating (DUB) enzymes free covalently linked ubiquitin moieties from ubiquitin-ubiquitin and ubiquitin-protein conjugates, and thereby maintain the equilibrium between free and conjugated ubiquitin moieties and regulate ubiquitin-mediated cellular processes. Here, we performed genetic analyses of mutant phenotypes in Drosophila melanogaster and demonstrate that loss of Usp14 function results in male sterility, with defects in spermatid individualization and reduced testicular free monoubiquitin levels. These phenotypes were rescued by germline-specific overexpression of wild-type Usp14. Synergistic genetic interactions with Ubi-p63E and cycloheximide sensitivity suggest that ubiquitin shortage is a primary cause of male sterility. In addition, Usp14 is predominantly expressed in testes in Drosophila, indicating a higher demand for this DUB in testes that is also reflected by testis-specific loss-of-function Usp14 phenotypes. Collectively, these results suggest a major role of Usp14 in maintaining normal steady state free monoubiquitin levels during the later stages of Drosophila spermatogenesis.This article has an associated First Person interview with the first author of the paper.

Developmental and tissue specific changes of ubiquitin forms in Drosophila melanogaster.

In most Eukaryotes, ubiquitin either exists as free monoubiquitin or as a molecule that is covalently linked to other proteins. These two forms cycle between each other and due to the concerted antagonistic activity of ubiquitylating and deubiquitylating enzymes, an intracellular ubiquitin equilibrium is maintained that is essential for normal biological function. However, measuring the level and ratio of these forms of ubiquitin has been difficult and time consuming. In this paper, we have adapted a simple immunoblotting technique to monitor ubiquitin content and equilibrium dynamics in different developmental stages and tissues of Drosophila. Our data show that the level of total ubiquitin is distinct in different developmental stages, lowest at the larval-pupal transition and in three days old adult males, and highest in first instar larvae. Interestingly, the ratio of free mono-ubiquitin remains within 30-50% range of the total throughout larval development, but peaks to 70-80% at the larval-pupal and the pupal-adult transitions. It stays within the 70-80% range in adults. In developmentally and physiologically active tissues, the ratio of free ubiquitin is similarly high, most likely reflecting a high demand for ubiquitin availability. We also used this method to demonstrate the disruption of the finely tuned ubiquitin equilibrium by the abolition of proteasome function or the housekeeping deubiquitylase, Usp5. Our data support the notion that the ubiquitin equilibrium is regulated by tissue- and developmental stage-specific mechanisms.

The Expression of Checkpoint and DNA Repair Genes in Head and Neck Cancer as Possible Predictive Factors.

DNA damage response failure may influence the efficacy of DNA-damaging treatments. We determined the expression of 16 genes involved in distinct DNA damage response pathways, in association with the response to standard therapy. Twenty patients with locoregionally advanced, squamous cell head and neck carcinoma were enrolled. The treatment included induction chemotherapy (iChT) with docetaxel, cisplatin and 5-fluorouracil followed by concomitant chemoradiotherapy (ChRT) or radiotherapy (RT) alone. The volumetric metabolic therapeutic response was determined by [18F]FDG-PET/CT. In the tumor and matched normal tissues collected before treatment, the gene expressions were examined via the quantitative real-time polymerase chain reaction (qRT-PCR). The down-regulation of TP53 was apparently associated with a poor response to iChT, its up-regulation with complete regression in 2 cases. 7 cases with down-regulated REV1 expression showed complete regression after ChRT/RT, while 1 case with REV1 overexpression was resistant to RT. The overexpression of WRN was an independent predictor of tumor relapse. Our results suggest that an altered expression of REV1 predicts sensitivity to RT, while WRN overexpression is an unfavorable prognostic factor.

Role of the deubiquitylating enzyme DmUsp5 in coupling ubiquitin equilibrium to development and apoptosis in Drosophila melanogaster.

Protein ubiquitylation is a dynamic process that affects the function and stability of proteins and controls essential cellular processes ranging from cell proliferation to cell death. This process is regulated through the balanced action of E3 ubiquitin ligases and deubiquitylating enzymes (DUB) which conjugate ubiquitins to, and remove them from target proteins, respectively. Our genetic analysis has revealed that the deubiquitylating enzyme DmUsp5 is required for maintenance of the ubiquitin equilibrium, cell survival and normal development in Drosophila. Loss of the DmUsp5 function leads to late larval lethality accompanied by the induction of apoptosis. Detailed analyses at a cellular level demonstrated that DmUsp5 mutants carry multiple abnormalities, including a drop in the free monoubiquitin level, the excessive accumulation of free polyubiquitins, polyubiquitylated proteins and subunits of the 26S proteasome. A shortage in free ubiquitins results in the induction of a ubiquitin stress response previously described only in the unicellular budding yeast. It is characterized by the induction of the proteasome-associated deubiquitylase DmUsp14 and sensitivity to cycloheximide. Removal of DmUsp5 also activates the pro-apoptotic machinery thereby resulting in widespread apoptosis, indicative of an anti-apoptotic role of DmUsp5. Collectively, the pleiotropic effects of a loss of DmUsp5 function can be explained in terms of the existence of a limited pool of free monoubiquitins which makes the ubiquitin-dependent processes mutually interdependent.

A novel interplay between the ubiquitin­proteasome system and serine proteases during Drosophila development.

The concentrations of the Drosophila proteasomal and extraproteasomal polyubiquitin receptors fluctuate in a developmentally regulated fashion. This fluctuation is generated by a previously unidentified proteolytic activity. In the present paper, we describe the purification, identification and characterization of this protease (endoproteinase I). Its expression increases sharply at the L1-L2 larval stages, remains high until the second half of the L3 stage, then declines dramatically. This sharp decrease coincides precisely with the increase of polyubiquitin receptor concentrations in late L3 larvae, which suggests a tight developmental co-regulation. RNAi-induced down-regulation of endoproteinase I results in pupal lethality. Interestingly, we found a cross-talk between the 26S proteasome and this larval protease: transgenic overexpression of the in vivo target of endoproteinase I, the C-terminal half of the proteasomal polyubiquitin receptor subunit p54/Rpn10 results in transcriptional down-regulation of endoproteinase I and consequently a lower level of proteolytic elimination of the polyubiquitin receptors. Another larval protease, Jonah65A-IV, which degrades only unfolded proteins and exhibits similar cross-talk with the proteasome has also been purified and characterized. It may prevent the accumulation of polyubiquitylated proteins in larvae contrary to the low polyubiquitin receptor concentration.

CalpB modulates border cell migration in Drosophila egg chambers.

BACKGROUND: Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility. RESULTS: We demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if), ß-PS integrin (mys) and talin (rhea) are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-head(R367A), a mutant form which is not able to bind ß-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts. CONCLUSIONS: The physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration.

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2.

BACKGROUND: Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C) plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. Here, lemming (lmg) mutants were used to study the APC/C subunit, Apc11, and its interaction partners in Drosophila melanogaster. RESULTS: The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg03424 P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the lmg138 null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite being conserved among Drosophila species, the LmgB protein is not required for viability or fertility. CONCLUSIONS: Our work provides insight into the subunit structure of the Drosophila APC/C with implications for its function. Based on the presented data, we suggest that the Lmg/Apc11 subunit recruits the E2-C type ubiquitin conjugating enzyme, Vihar, to the APC/C together with Mr/Apc2 by forming a ternary complex.

Overexpression of Dsk2/dUbqln results in severe developmental defects and lethality in Drosophila melanogaster that can be rescued by overexpression of the p54/Rpn10/S5a proteasomal subunit.

Polyubiquitin receptors execute the targeting of polyubiquitylated proteins to the 26S proteasome. In vitro studies indicate that disturbance of the physiological balance among different receptor proteins impairs the proteasomal degradation of polyubiquitylated proteins. To study the physiological consequences of shifting the in vivo equilibrium between the p54/Rpn10 proteasomal and the Dsk2/dUbqln extraproteasomal polyubiquitin receptors, transgenic Drosophila lines were constructed in which the overexpression or RNA interference-mediated silencing of these receptors can be induced. Flies overexpressing Flag-p54 were viable and fertile, without any detectable morphological abnormalities, although detectable accumulation of polyubiquitylated proteins demonstrated a certain level of proteolytic disturbance. Flag-p54 was assembled into the 26S proteasome and could fully complement the lethal phenotype of a p54 null mutant Drosophila line. The overexpression of Dsk2 caused severe morphological abnormalities in the late pupal stages, leading to pharate adult lethality, accompanied by a huge accumulation of highly polyubiquitylated proteins. The lethal phenotype of Dsk2 overexpression could be rescued in a double transgenic line coexpressing Flag-Dsk2 and Flag-p54. Although the double transgenic line was viable and fertile, it did not restore the proteolytic defects; the accumulation of the highly polyubiquitylated proteins was even more severe in the double transgenic line. Significant differences were found in the Dsk2-26S proteasome interaction in Drosophila melanogaster as compared with Saccharomyces cerevisiae. In yeast, Dsk2 can interact only with ΔRpn10 proteasomes and not with the wild-type one. In Drosophila, Dsk2 does not interact with Δp54 proteasomes, but the interaction can be fully restored by complementing the Δp54 deletion with Flag-p54.

Developmental-stage-specific regulation of the polyubiquitin receptors in Drosophila melanogaster.

Recognition of polyubiquitylated substrates by the proteasome is a highly regulated process that requires polyubiquitin receptors. We show here that the concentrations of the proteasomal and extraproteasomal polyubiquitin receptors change in a developmentally regulated fashion. The stoichiometry of the proteasomal p54/Rpn10 polyubiquitin receptor subunit, relative to that of other regulatory particle (RP) subunits falls suddenly at the end of embryogenesis, remains low throughout the larval stages, starts to increase again in the late third instar larvae and remains high in the pupae, adults and embryos. A similar developmentally regulated fluctuation was observed in the concentrations of the Rad23 and Dsk2 extraproteasomal polyubiquitin receptors. Depletion of the polyubiquitin receptors at the end of embryogenesis is due to the emergence of a developmentally regulated selective proteolytic activity. To follow the fate of subunit p54/Rpn10 in vivo, transgenic Drosophila melanogaster lines encoding the N-terminal half (NTH), the C-terminal half (CTH) or the full-length p54/Rpn10 subunit were established in the inducible Gal4-UAS system. The daughterless-Gal4-driven whole-body expression of the full-length subunit or its NTH did not produce any detectable phenotypic changes, and the transgenic products were incorporated into the 26S proteasome. The transgene-encoded CTH was not incorporated into the 26S proteasome, caused third instar larval lethality and was found to be multi-ubiquitylated. This modification, however, did not appear to be a degradation signal because the half-life of the CTH was over 48 hours. Accumulation of the CTH disturbed the developmentally regulated changes in subunit composition of the RP and the emergence of the selective proteolytic activity responsible for the depletion of the polyubiquitin receptors. Build-up of subunit p54/Rpn10 in the RP had already started in 84-hour-old larvae and reached the full complement characteristic of the non-larval developmental stages at the middle of the third instar larval stage, just before these larvae perished. Similar shifts were observed in the concentrations of the Rad23 and Dsk2 polyubiquitin receptors. The postsynthetic modification of CTH might be essential for this developmental regulation, or it might regulate an essential extraproteasomal function(s) of subunit p54/Rpn10 that is disturbed by the expression of an excess of CTH.

APC/CFzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle.

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. Here, we report that APC/C Fzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. We demonstrate that compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, our data suggest that the activity of APC/C Fzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, our data suggest a model in which the cyclic activity of APC/C Fzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. We propose that, as is observed in mitotic cycles, during endocycles, APC/C Fzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression.